A biocompatible, non-biodegradable, non-toxic polymer useful for nanoparticle pharmaceutical compositions

ABSTRACT

The invention relates to a biocompatible, non-biodegradable, and non-toxic polymer of formula (I), comprising of three monomeric units, selected from 1-Vinylpyrrolidone (VP), N-Isopropylacrylamide (NIPAM), and ester of Maleic anhydride and Polyethylene glycol (MPEG), cross-linked with a bi-functional vinyl derivative, of high purity and substantially free of respective toxic monomeric contaminants, and a process for preparation thereof. The invention further relates to nanoparticulate pharmaceutical compositions of poorly water-soluble drugs or compounds comprising the polymer of the invention, which are safe, less-toxic and convenient for bedside administration to patients in need thereof. Furthermore, the invention relates to a highly selective method for preparation of nanoparticulate pharmaceutical compositions of poorly water-soluble drugs or compounds.

FIELD OF THE INVENTION

The present invention provides a biocompatible and non-biodegradablepolymer of high purity, substantially free of monomeric contaminants anda process for preparation thereof.

The present invention further provides pharmaceutical compositions ofpoorly water-soluble drugs or compounds in nanoparticulate formutilizing the said polymer of the invention.

Furthermore, the present invention relates to a highly selective methodfor preparation of pharmaceutical compositions of poorly water-solubledrugs or compounds in nanoparticulate form as well as a method ofadministration of the same to patients in need thereof.

The polymer of the present invention being non-toxic and safe, therebyrender the nanoparticulate pharmaceutical compositions of poorlywater-soluble drugs or compounds comprising the said polymer alsoless-toxic and safer for administration.

BACKGROUND OF THE INVENTION

Recent years have seen an ever-increasing interest in the application ofnovel materials in the medical and pharmaceutical fields, whether asprostheses or in medical devices designed for contact with biologicalenvironment of the living body. Of these materials, polymers, mainlysynthetic polymers, are by far the most diverse classes that are foundto impart considerable benefits to the patient health care.

The applications of polymers in the medical and pharmaceutical field arewide ranging. In the medical field, polymers are employed as implants orsupport materials such as artificial organs, vascular grafts,intraocular lenses, artificial joints, mammary prostheses, suturematerials, extracorporeal therapeutics or other support materials suchas those used in hemoperfusion, blood oxygenators, catheters, bloodtubing, wound and burn covering materials, splints, contact lenses etc.In the pharmaceutical field, polymers have been particularly used indevelopment of nanoparticle delivery systems and controlled-releasedelivery systems. Extensive studies are also being pursued to targetdrugs with delivery systems to the desired site. Further, polymers havefound great utility in other applications as well, such as transdermaldrug-delivery patches, micro spheres, bioprocesses such as enzyme andcell immobilization etc.

Amongst such applications, nanoparticulate drug delivery systems havebeen more extensively studied and nanometer-size drug carriers withhydrophilic surfaces, specially those comprising two sphericalco-centric regions of polymeric micelles—a densely packed core of ahydrophobic material, which is responsible for entrapping a hydrophobicdrug or compound and an outer shell made of hydrophilic material havebeen extensively studied. Such systems are found to evade recognitionand uptake by the reticulo-endothelial systems (RES) and thus cancirculate in the blood for a long time. Further, due to their extremelysmall size (a polymeric micelle usually consists of several hundredblock copolymers and has a diameter of about 20 nm-50 nm), the particlesextravasate at the pathological sites, such as solid tumors throughpassive targeting mechanism.

Polymers derive their range of properties attributable to their chemicaland structural features. The polymer chains may essentially be linear,branched, or cross-linked to adjacent chains. Furthermore, these chainsmay be unordered, ordered or oriented in a single direction. Thesestructural features combined with the chemical composition, lends avariety of properties to polymers, resulting in a variety of end-useapplications. Further, these structural features combined with thechemical composition may impart or deprive the resultant polymer,biocompatibility and resistance to biodegradation by the host tissueenvironment. These factors also influence other properties such assolubility and methods of processing and moulding.

Moreover, when a polymer is injected into the mammals, it normally,slowly disappears from the site of administration, however, thisdisappearance occurs in response to a chemical reaction such ashydrolysis, which normally is a part of biotransformation process andthe said polymer is metabolised and eliminated from the body. This,however, sometimes leads to unnecessary metabolites, which causeuntoward effects on various biological systems. Therefore, polymers,which are inert in/to the environment of use, and are eliminated orextracted intact from the site of administration as well as serveessentially as a rate limiting barrier to transport and release of adrug from it, may be of prime importance based on the intendedfunctions. Again, the biodegradability of a polymer depends on themechanical and chemical properties of the polymer. A variety of natural,synthetic, and biosynthetic polymers are bio- and environmentallydegradable. A polymer based on the C—C backbone tends to benon-biodegradable, whereas hetero atom-containing polymer backbonesconfer biodegradability. Non-biodegradability/biodegradability cantherefore be engineered into polymers by the judicious deletion/additionof chemical linkages such as anhydride, ester, or amide bonds, amongothers Common examples of non-biodegradable polymer materials includepolyethylene vinyl acetate, polydimethyl siloxane, polyether urethane,ethyl cellulose, cellulose acetate, polyethylene and polyvinyl chloride.

There is a welter of reports available on the attempts made over thelast few decades or so on development of nanoparticulate deliverysystems for a large variety of drugs utilizing polymers. To name a few,these include the disclosures of:

-   i) Sakurai et al in U.S. Pat. No. 5,412,072, wherein a complex    comprising a drug covalently bonded to a polymer composed of    hydrophilic and hydrophobic fragments is found to render the said    complex water soluble and thereby suitable for administration. The    drugs utilized therein are in general less water soluble or    insoluble compounds and the drug-polymer complex is reported to form    polymeric micelles in aqueous solutions and becomes water-soluble    high molecular polymerized drugs, useful and suitable for    administration.-   ii) Yokoyama et al in U.S. Pat. No. 5,449,513, wherein they report a    polymeric micelle, which unlike that disclosed by Sakurai et al in    U.S. Pat. No. 5,412,072 is not a complex wherein a drug is    covalently bonded to a polymer, but rather one wherein the drug is    entrapped within the polymer. The drugs utilized for entrapment are    hydrophobic in nature. The polymeric micelle is in turn prepared by    entrapment of hydrophobic drugs inside the polymeric shell through    conventional methods such as ultra sonication, followed by    purification of the micelles thus obtained through dialysis.-   iii) Desai et al in U.S. Pat. No. 5,439,686; U.S. Pat. No.    5,362,478; U.S. Pat. No. 5,916,596; U.S. Pat. No. 6,096,331; U.S.    Pat. No. 6,537,579 and U.S. Pat. No. 6,749,868, wherein polymeric    micelle of substantially water-insoluble compounds are prepared. The    water-insoluble compound is reported to be entrapped inside the    polymeric shell to a significant extent and suitable for    administration to a patient in need thereof either in a soluble or    suspended form.

The polymers utilized by Sakurai et al in U.S. Pat. No. 5,412,072 are ingeneral those comprising a hydrophilic segment selected frompolyethylene glycol, polysaccharides, polyacrylamide etc and ahydrophobic segment selected from polyaspartic acid, polyglutamic acid,polylysine etc.

The polymers utilized by Yokoyama et al in U.S. Pat. No. 5,449,513 arein general those comprising a hydrophilic segment selected frompolyethylene oxide, polymalic acid, polyaspartic acid, polyglutamicacid, polylysine, polysaccharide etc and a hydrophobic segment selectedfrom poly (β-benzyl L-aspartate), poly(γ-benzyl L-glutamate), poly(β-substituted aspartate), poly (γ-substituted glutamate),poly(L-leucine), poly(L-valine), poly(L-phenylalanine), hydrophobicpolyamino acids, polystyrene, polymethacrylate, polyacrylate,polymethacrylate amide, polyacrylate amide, polyamide, polyester,polyalkylene oxide and hydrophobic polyolefins.

The polymers utilized by Desai et al in U.S. Pat. No. 5,439,686; U.S.Pat. No. 5,362,478; U.S. Pat. No. 5,916,596; U.S. Pat. No. 6,096,331;U.S. Pat. No. 6,537,579 and U.S. Pat. No. 6,749,868 are in general thoseessentially bearing sulfhydryl groups or disulfide bonds within itsstructure e.g. Albumin (which contains 35 cysteine residues), Insulin(which contains 6 cysteine residues), Haemoglobin (which contains 6cysteine residues per α₂ β₂ unit), Lysozyme (which contains 8 cysteineresidues), Immunoglobulins, α-2-Macroglobulin, Vitronectin, Vitronectin,Fibrinogen etc. Such polymers are substantially cross-linked throughformation of disulphide bonds. Such polymers include both synthetic andnatural polymers, which as mentioned hereinbefore, bear sulfhydrylgroups or disulfide bonds within their structure. The sulfhydryl groupsor disulfide linkages are reported to either be pre-existing or obtainedthrough suitable chemical modifications. The natural polymers arereported to be preferred and include albumin proteins, oligopeptides,polynucleic acids etc.

However, the disadvantage with the polymeric micelles disclosed bySakurai et al, Yokoyama et al, and Desai et al are that they all utilizepolymers, both synthetic and natural, which are biodegradable. It mightbe mentioned that biodegradable polymers, although, are capable ofinfluencing the drug release pattern as well as the release kinetics ofthe loaded drug, however, are not particularly preferred in drugdelivery systems because they:

-   a) Have low plasma life time due to their rapid capture by the    mononuclear phagocyte system (MPS) cells;-   b) Lack response to physiological changes;-   c) Lack consistent drug release kinetics which may economically and    therapeutically cause waste of the drugs and other adverse effects;    and-   d) May cause increase of toxicity or immunogenicity since    encapsulation of protein drugs involves organic solvents, which may    cause protein denaturation.

Delivery systems wherein polymers, which are non-biodegradable, havebeen utilized and disclosed by:

-   i) Maitra et al in U.S. Pat. No. 5,874,111, wherein the drug is    entrapped within the polymer resulting in a highly monodispersed    polymeric hydrophilic nanoparticle being formed. The polymers    utilized are those comprising of monomers like Vinylpyrrolidone (VP)    or mixture of Vinylpyrrolidone and polyethyleneglycolfumarate    (PEGF), etc.-   ii) Maitra et al in U.S. Pat. No. 6,322,817, wherein the polymers    utilized comprise of at least one type of an amphiphilic monomer    selected from the group consisting of vinylpyrrolidone, acrylic    acid, alkyl acrylates having a chain length of C₃ to C₆    functionalized polyethylene glycol of a molecular weight of 2000 to    6000, N-alkylacrylamide having a chain length of C₃ to C₆, and    alkylcyanoacrylate having a chain length of C₃ to C₆. The drugs    entrapped within the polymeric micelles are taxane derivatives, in    particular Paclitaxel.-   iii) Lowe et al in US 2005/0169882, wherein the polymers utilized    comprise of a smart segment (which is non-biodegradable) and a    biodegradable segment. More specifically, the smart,    non-biodegradable segment comprises of poly(N-isopropylacrylamide),    poly(N-alkylacrylamide), poly(N-n-propylacrylamide),    poly(N-isopropylmethacrylamide), poly(ethylene oxide)-poly(propylene    oxide)-poly(ethylene oxide), elastin-like polypeptides, or a    derivative thereof and the biodegradable segment comprises of    polysaccharide, dextran, polyester, polylactide, poly(L-lactic    acid), poly(D,L-lactic acid), poly(lactide-co-glycolides),    biotinylated poly(ethylene glycol-block-lactic acid) etc.

In the case of polymers disclosed in U.S. Pat. No. 5,874,111, it mightbe noted that such polymers are prepared through polymerization ofrespective monomers and the polymeric material thus obtained is purifiedand isolated from an aqueous medium containing the same through a methodof dialysis.

In the case of polymers disclosed in U.S. Pat. No. 6,322,817 also, thepolymers are prepared through polymerization of respective monomers andis purified and isolated from an aqueous medium through a method ofdialysis.

Similarly, the polymers disclosed by Lowe et al in US 2005/0169882 isalso purified and isolated from an aqueous medium containing the samethrough a method of dialysis.

With regard to the compounds or drugs disclosed by Maitra et al in U.S.Pat. No. 5,874,111 for entrapment into polymers disclosed therein, theyare primarily Antigens, Bovine serum etc. In the case of U.S. Pat. No.6,322,817, the drugs for entrapment into polymers disclosed therein areprimarily water-insoluble taxane derivatives, especially Paclitaxel,whereas Lowe et al in US 2005/0169882 disclose a wide host of drugs,which can be entrapped into the polymeric shell disclosed therein.

Further to the above, Burman et al in U.S. Pat. No. 6,365,191 teach apharmaceutical composition comprising the polymer disclosed in U.S. Pat.No. 6,322,817, and more specifically a pharmaceutical composition oftaxane derivatives, especially Paclitaxel. Herein, the saidpharmaceutical composition is prepared by adding a solution ofPaclitaxel in ethanol to an infusion vehicle comprising dextrosesolution, to which has been added a solution of polymer in watercontaining an anionic surfactant and a buffering agent. Burman et alfurther claim that the pharmaceutical composition is stable for morethan 12 hours without any precipitation of the drug from the perfusionfluid and that more than 90% (as analysed by HPLC) of the drug isentrapped within the polymeric micelle even after 24 hours ofpreparation of the perfusion fluid.

Even though, Burman et al in U.S. Pat. No. 6,365,191 claim that thepharmaceutical composition disclosed therein is in a nanoparticulateform, however, there is no mention in the specification as to the sizeof the claimed nanoparticulate form. The only mention about the particlesize of the polymeric micelles containing Paclitaxel can be found in thedisclosure by Maitra et al in U.S. Pat. No. 6,322,817, wherein the saidnanoparticles containing Paclitaxel are reported to have a diameter inthe range of 30-1501 nm. Similarly, there is no mention about particlesize of the polymeric micelles of the compositions disclosed by Lowe etal in US 2005/0169882.

It is important to note that the polymers disclosed in U.S. Pat. No.5,874,111; U.S. Pat. No. 6,322,817; U.S. Pat. No. 6,365,191 and US2005/0169882 are prepared from one or more monomers, which includeVinylpyrrolidone and N-Isopropylacrylamide. It is further, important tonote that Vinylpyrrolidone and N-Isopropylacrylamide are toxiccompounds, whose presence in a pharmaceutical composition meant forhuman/animal consumption is not only frowned upon by Health Authoritiesworldover, but also comes under stringent quality adherence, with strictlimits set by Pharmacopoeial Forums worldover. For instance, the levelof monomeric Vinylpyrrolidone in the polymer, Polyvinylpyrrolidone, aswell as any other polymer containing Vinylpyrrolidone as a monomer, asprescribed by US and European Pharmacopoeias should not exceed a limitof 0.001% (i.e. <10 ppm).

There is a grave danger that in the methods described in U.S. Pat. No.5,874,111; U.S. Pat. No. 6,322,817; U.S. Pat. No. 6,365,191 and US2005/0169882 for preparation and isolation of polymers utilizingVinylpyrrolidone as one of the monomers, the said monomer i.e.Vinylpyrrolidone may be present in limits higher than 0.001% (i.e. >10ppm).

There is an equally grave danger that pharmaceutical compositionscontaining such polymers, utilising Vinylpyrrolidone as one of themonomers may also contain Vinylpyrrolidone as a monomeric contaminantand that the said monomer i.e. Vinylpyrrolidone may also be present inlimits higher than 0.001% (i.e. >10 ppm).

This could be true especially with regard to the pharmaceuticalcompositions disclosed in U.S. Pat. No. 6,322,817; U.S. Pat. No.6,365,191 and US 2005/0169882.

It need not be overemphasised that any chemical reaction including areaction for preparation of polymers is never complete in the sense thatinvariably one or more of the reactants are left over in the product.This would apply to polymerization reactions involving Vinylpyrrolidoneas a monomer and depending on the molar or weight proportions ofVinylpyrrolidone utilized, there is every possibility that some amountof the Vinylpyrrolidone would remain as a contaminant in the polymericproducts prepared thereof.

In connection with the above, the present inventors concerns were foundtrue, wherein analysis of the polymer prepared by polymerization ofthree monomers, viz. Vinylpyrrolidone (VP), N-isopropylacrylamide(NIPAM) and Ester of Maleic anhydride and polyethylene glycol (MPEG)exactly as per the description given in Examples I, II and III of U.S.Pat. No. 6,322,817 were found to contain an amount ofN-isopropylacrylamide (NIPAM) and Vinylpyrrolidone (VP) as summarizedTable-I.

TABLE I Amount of Residual Monomers In The Polymer Prepared As Per TheMethod Described In Examples I, II and III Of U.S. Pat. No. 6,322,817Monomer % w/w Detected In The Polymer NIPAM 0.066-0.076 (660-760 ppm) VP0.008-0.011 (80-110 ppm)

It would be abundantly evident that the amount of Vinylpyrrolidone foundin the polymer is at least more than eight times the toxic limit of0.001% (i.e. 10 ppm), wherein the pharmaceutical composition containingsuch a polymer would be very unsafe and highly toxic for administrationto humans or animals.

A need, if not imperative exists not only for a polymer substantiallyfree of toxic monomers, such as N-isopropylacrylamide (NIPAM) andVinylpyrrolidone (VP) but also for pharmaceutical compositionscomprising a polymer, which are substantially free of toxic monomericcontaminants, such as N-isopropylacrylamide (NIPAM) and Vinylpyrrolidone(VP).

It might also be noted that the pharmaceutical compositions disclosed byBurman et al in U.S. Pat. No. 6,365,191 are reported to have a stabilityof only about 12 hrs or more with 90% or more of the drug entrappedwithin the polymeric micelle at the end of 24 hours. Further, thepharmaceutical compositions disclosed by Lowe et al in US 2005/0169882are reported to have a loading of the biologically active substances ofapproximately 40% only, with a release of the biologically activesubstance between a few hours to up to several days.

A further need exists for pharmaceutical compositions, which have longerstability as well as higher drug loading, which moreover are safe andless-toxic.

The present invention is a step forward in advancement of not onlyproviding a polymer, which is substantially free of toxic monomericcontaminants, but also providing a pharmaceutical composition comprisingsuch a desired polymer, which is safe for human/animal administrationand has longer stability.

OBJECTS OF THE INVENTION

An object of the present invention is to provide a biocompatible andnon-biodegradable polymer of high purity, substantially free ofmonomeric contaminants.

Another object of the present invention is to provide a process forpreparation of a biocompatible and non-biodegradable polymer of highpurity, substantially free of monomeric contaminants.

Yet another object of the present invention is to provide pharmaceuticalcompositions of poorly water-soluble drugs or compounds innanoparticulate form comprising biocompatible and non-biodegradablepolymers, of high purity and substantially free of monomericcontaminants.

A further object of the present invention is to provide a method forpreparation of pharmaceutical compositions of poorly water-soluble drugsor compounds in nanoparticulate form utilizing biocompatible andnon-biodegradable polymers, of high purity and substantially free ofmonomeric contaminants.

Yet further object of the present invention is to providenanoparticulate pharmaceutical compositions of poorly water-solubledrugs or compounds comprising biocompatible and non-biodegradablepolymers of high purity and free of monomeric contaminants, which aresafe and less-toxic.

Another objective of the present invention is to provide a highlyselective method for preparation of pharmaceutical compositions innanoparticulate form comprising poorly water soluble drugs or compoundsand a biocompatible and non-biodegradable polymer of high purity andfree of monomeric contaminants.

Yet another object of the present invention is to provide a method ofadministration of the nanoparticulate pharmaceutical compositionscomprising a biocompatible and non-biodegradable polymer of highlypurity and substantially free of monomeric contaminants to patients inneed thereof.

A further object of the present invention is to provide nanoparticulatepharmaceutical compositions comprising a biocompatible andnon-biodegradable polymer of high purity and free of monomericcontaminants, which are stable for longer periods of time.

Abbreviations Used in the Invention VP=Vinylpyrrolidone or1-Vinylpyrrolidin-2-one NIPAM=N-Isopropylacrylamide

MPEG=Ester of Maleic anhydride and Polyethylene glycolMBA=N,N′-methylenebisacrylamide

TMED=Tetramethylethylenediamine

APS=Ammonium persulphate

LCST=Lower Critical Solution Temperature DSC=Differential ScanningCalorimetry TGA=Thermogravimetric Analysis CMC=Critical MicelleConcentration

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The ¹H-NMR Spectrum of the Polymer of the present invention.

FIG. 2: The ¹³C-NMR Spectrum of the Polymer of the present invention.

FIG. 3: The Fourier Transform Infrared (FT-IR) Spectrum of the Polymerof the present invention.

FIG. 4: The TGA Thermogram of the Polymer of the present invention.

FIG. 5: The DSC Thermogram of the Polymer of the present invention.

FIG. 6: The Pharmacokinetic Blood Profile of the [¹⁴C]-labelled Polymerof the present invention.

FIG. 7: Photographs of S&E stained Rabbit Ear Lobe site after 48 hoursof the subcutaneous administration of a 10% Dextrose solution (Control).

FIG. 8: Photographs of S&E stained Rabbit Ear Lobe site after 48 hoursof the subcutaneous administration of an aqueous solution of the Polymerof the present invention.

FIG. 9: Photographs of S&E stained Rabbit's Marginal Ear Vein site after24 hours of the Intravenous administration of a 10% Dextrose solution(Control).

FIG. 10: Photographs of S&E stained Rabbit's Marginal Ear Vein siteafter 24 hours of the Intravenous administration of an aqueous solutionof the Polymer of the present invention.

FIG. 11: A Representation of a typical method for preparation andadministration to patients in need thereof of the Nan particulatePharmaceutical Composition of the present invention.

SUMMARY OF THE INVENTION

In their endeavours, the present inventors have found that all theobjectives set forth could be achieved, which moreover overcomes most,if not all the limitations of the prior art.

In the first place, the present inventors have found that a polymercomprising a monomeric unit of both NIPAM and VP could be obtained,wherein the amount of the respective individual monomeric contaminantsin the polymer thus obtained is below the toxic limits prescribed.

In particular, the present inventors have found that a polymercomprising NIPAM, VP and MPEG as monomeric units could be obtained,which is substantially free of monomeric contaminants of toxic NIPAM andVP. Specifically, the present inventors have found that such a polymercould be obtained in high purity containing the said NIPAM and VPcontaminants in levels much lower than 0.001% w/w.

The polymer having the desired characteristics could be obtained througha highly selective method, comprising subjecting an aqueous solutioncontaining the polymer obtained by polymerization of NIPAM, VP and MPEGto a step of diafiltration or ultrafiltration, unlike the methods ofdialysis, as taught in the prior art.

That the polymer obtained by the method of the present invention issuperior to prior art methods, especially the method disclosed in U.S.Pat. No. 6,322,817, involving a step of dialysis could be bestunderstood from a comparison of the residual monomers contained in therespective polymers, as summarized in Table-II.

TABLE II A Comparison Of The Monomeric Contaminants Contained In ThePolymer Obtained As Per The Process Disclosed in U.S. Pat. No.6,322,817, Utilising Dialysis Vis-à-vis The Polymer Obtained As Per ThePresent Invention, Utilising Diafiltration The Polymer Obtained As PerThe The Polymer Obtained As Per The Parameter Method Of U.S. Pat. No.6,322,817 Method Of The Present Invention Residual NIPAM 0.066-0.076%w/w <0.001% w/w (660-760 ppm) (<10 ppm) Residual VP 0.008-0.011% w/w<0.001% w/w (80-110 ppm) (<10 ppm)

It should be noted that dialysis is a process, which involves slowseparation of smaller molecules from larger molecules or of dissolvedsubstances from colloidal particles in a solution by selective diffusionthrough a semipermeable membrane. In a typical dialysis method, thepolymer solution for purification is contained within a semipermeablemembrane and the low-molecular weight solutes (monomers) are removed byplacing a pure solvent, generally water outside the membrane. Thissolvent is changed periodically or continuously until the concentrationof diffusible solutes (monomers like NIPAM and VP) in the solutioncontaining the polymer is reduced.

Unlike the dialysis method, the diafiltration technique involvesmechanical flow of fluid by a pump across the membrane, whereby thefluid is pumped tangentially (for this reason diafiltration is alsoknown as Tangential Flow Filtration), along the surface of the membrane.An applied pressure forces a portion of the fluid to diffuse selectivelythrough the membrane to the filtrate side. The retained polymericcomponents do not build up at the surface of the semipermeable membrane.

In contrast to the dialysis method, wherein the initial quantity orvolume of the aqueous solution of the polymer taken up for purificationremains as such/or same or gets diluted at the end or completion of thesaid operation, in the diafiltration method, on the other hand, sincethe aqueous solution of the polymer is swept along by tangential flow,concurrently results in concentration of the said solution containingthe polymer, resulting in a more concentrated solution of the polymer.For example, when a solution containing, say 100 gm of the polymer in5.0 Lts of water is subjected to dialysis and diafiltrationrespectively, at the end of the former method a solution containing 100gm of the polymer in 4.5 to 5.5 Lts of water is obtained, whereas at theend of the latter method a reduction in or concentration of the initialvolume results, generally to a tune of between one-fourth to one-sixth(¼^(th) to ⅙^(th)) the initial volume, and at the end a solution of 100gm of the polymer in about 0.8 to 1.3 Lts of water is obtained.

Further, as mentioned hereinbefore, in the dialysis method the solventkept outside the membrane containing the solution of the polymer needsto be changed periodically or continuously manually, whereas in thediafiltration method no such manual operation of periodic or continuouschange of any solvent is necessary. This renders the former tediousrequiring manual supervision during its operation, whereas the lattermethod is fully automated and regulated and hence less tedious andconvenient requiring no manual supervision during its operation.

Furthermore, by virtue of the diafiltration method being operable undera closed environment in comparison to the dialysis method, whichoperates under an open environment, any possibility of microbialcontamination is minimal or non-existent in the former method andtherefore, would qualify as an operation under aseptic conditions,conforming to not only Good Manufacturing Practices but also conformingto Regulatory Guidelines worldover.

Further, by virtue of not only involving manual operation but also theinherent principle tinder which the dialysis method works, the timecycle for an unit operation is lengthy and generally takes about 24-36hours. In comparison, the diafiltration method by virtue of the inherentprinciple under which it works requires a very short time cycle andusually takes less than one hour for completion. To say in other words,the diafiltration method is at least twenty five (25) times faster thanthe dialysis method and hence industrially more suited.

While, the dialysis method calls for a rather lengthy time cycle foroperation and completion, another inherent disadvantage of the saidmethod is that it allows only a small volume/quantity of a solution ofthe polymer to be purified. Such limitations do not rest with thediafiltration method and in general it allows a larger volume/quantity,say to a tune of at least 4 to 5 times of a solution to be purified. Forexample, if the dialysis method has a capacity for purifying a solutionof 100 gm of the polymer in 5.0 Lts of water in one cycle, by thediafiltration method in one cycle between 400 to 500 gm of the polymerin 20.0 to 25.0 Lts of water can be purified. Needless to mention, thelarger processing capacity of the diafiltration method is anotheradvantage it offers in addition to the others already discussedhereinbefore i.e. faster operation time, automated and regulatedoperation, convenient and less tedious, concurrent concentration of orreduction in volume of the initial solution, conforming to GoodManufacturing Practices and Regulatory Guidelines, minimal ornon-existent microbial contamination etc.

However, as mentioned and discussed hereinbefore, the most importantadvantage the diafiltration method has over the dialysis method is inthe purity of the polymer obtained by the respective methods, with theformer method producing the polymer, substantially free of monomericcontaminants and hence non-toxic and safe for human/animal consumption[Refer Table-II].

Last, but not the least, another inherent advantage of the diafiltrationmethod is that if the polymer is required/necessitated to be isolated insolid form from the solution, through evaporation of the solvent, e.g.through lyophilization then by virtue of the said method resulting in areduction in or concentration of the initial volume of the polymersolution, generally to a tune of about one fourth to one-sixth (¼^(th)to ⅙^(th)) of the initial volume, the evaporation/lyophilization timecycle would also be drastically reduced. In the dialysis method, sinceno reduction in or concentration of the initial volume takes place, ifthe polymer is required/necessitated to be isolated in solid form fromthe solution, the evaporation/lyophilization time cycle would be muchhigher.

In short, the diafiltration method in comparison to the dialysis methodis easily scaleable for large manufacturing, conforming to GoodManufacturing Practices, and hence industrially more viable andfriendly.

The essential differences between the diafiltration and the dialysismethods in are summarised in Table-III, for quick reference.

While, to some extent the diafiltration or ultrafiltration technique isapplied to various purifications, however, their application in thefield of polymers is hitherto not reported and forms the novel andinventive aspect of the present invention.

Further, the present inventors have further characterized the polymer ofhigh purity and substantially free from monomeric contaminants thusobtained, by various spectroscopic methods such as ¹H-NMR, ¹³C-NMR andFourier Transform Infrared (FT-IR) and found it to have the structure(I) as shown hereinbelow:

Furthermore, based on studies with Radio-labelled polymer in male Swissalbino mice, as would be evident from the details given in the laterpart of this Specification [refer Tables-VI and VII and FIG. 6], it wasfound that the polymer of the present invention is non-biodegradable andis rapidly eliminated from the body without being deposited and degradedin vital organs, suggesting the safety and utility of the polymer forhuman/animal use.

TABLE III A Comparison Of The Dialysis And Diafiltration MethodsVis-à-vis Purification Of An Aqueous Solution Of The Polymer Of ThePresent Invention Parameter Dialysis Method Diafiltration Method BatchSize Limited and Hence a Constraint At least 4-5 times the (e.g. 100 gmof Polymer in 5.0 Volume/Quantity can be Processed Lts of water) (e.g.400 to 500 gm of Polymer in 20.0 to 25.0 Lts of water) ProcessingConditions Open Environment Closed Environment Processing Time Lengthy(24-36 Hours) Shorter (<1 Hour) Operation and Manual; Tedious; LessAutomated and Regulated; Less Convenience Convenient Tedious; HighlyConvenient Microbial High Possibility Minimal or Non-ExistentContamination Reduction None About one-fourth to one-sixthin/Concentration of (¼^(th) to ⅙ ^(th) ) Initial Volume of the SolutionLyophilization Time Lengthy (90-110% of Initial Shorter (only 15-25% ofInitial Cycle of The solution Volume to be Lyophilized) Volume to beLyophilized) Monomeric Very High Below Pharmacopoeial and ToxicContamination Limits Scalability and Difficult; Less Viable EasilyScaleable; Conforming to Industrial Viability Good ManufacturingPractices; Highly Viable

Further, based on extensive toxicity studies such as Localized Toxicity(Subcutaneous and Intravenous), Target Organs Dose Toxicity up to 800mg/kg animal body weight, Six Months Cyclical Dose Toxicity etc., aswould be evident from the details given in the later part of thisSpecification [refer FIGS. 7-10], it was found that the polymer of thepresent invention polymer is non-toxic, biocompatible and biologicallysafe for use in making pharmaceutical compositions meant forhuman/animal use and administration.

It was further found that the polymer of the present invention, which isof high purity and substantially free of monomeric contaminants andfurther being biocompatible, non-biodegradable, safe and non-toxic isparticularly useful in preparation of pharmaceutical compositions,comprising the said polymer in nanoparticulate form along withpharmaceutically acceptable excipients, which in turn are safe andless-toxic for human/animal use and/or administration.

Especially, the biocompatible, non-biodegradable, safe and non-toxicpolymer of the present invention was found to entrap completely ornear-completely within its polymeric shell a host of poorlywater-soluble drugs or compounds, wherein the said poorly water-solubledrugs or compounds are available in nanoparticulate form, in particlesizes of between 30-150 nm.

Structure of the Polymer of the Present Invention

In particular, it was found that the polymer of the present inventioncould be used to prepare a pharmaceutical composition in nanoparticulateforms, along with pharmaceutically acceptable excipients entrapping ahost of poorly water-soluble drugs or compounds completely ornear-completely within its polymeric shell. Such poorly water-solubledrugs or compounds are those having water solubility of less than 10mg/ml. Examples of such poorly water-soluble drugs or compounds include,but are not limited to, anticancer agents, anti-inflammatory agents,anti-fungal agents, antiemetics, antihypertensive agents, sex hormones,steroids, antibiotics, immunomodulators, anaesthetics etc. Typicalexamples of anticancer agents that can be entrapped within the polymericshell are Paclitaxel, Docetaxel, and other related taxane derivatives;Irinotecan, Topotecan, and other related Camptothecin derivatives;Doxorubicin, Daunomycin, and related Anthracycline derivatives;Cisplatin; Oxaliplatin; 5-Fluorouracil; Mitomycin; Methotrexate;Etoposide; Betulinic acid and its derivatives; and Wedelolactone and itsderivatives. Typical examples of anti-inflammatory agents that can beentrapped within the polymeric shell include Indomethacin, Ibuprofen,Ketoprofen, Flubiprofen, Piroxicam, Tenoxicam, and Naproxen. Typicalexamples of anti-fungal agents that can be entrapped within thepolymeric shell include Ketoconazole, and Amphotericin B. Typicalexamples of sex hormones that can be entrapped within the polymericshell include Testosterone, Estrogen, Progesterone, and Estradiol.Typical examples of steroids that can be entrapped within the polymericshell include Dexamethasone, Prednisolone, and Triamcinolone. Typicalexamples of antihypertensive agents that can be entrapped within thepolymeric shell include Captopril, Ramipril, Terazosin, Minoxidil, andParazosin. Typical examples of antiemetics that can be entrapped withinthe polymeric shell include Ondansetron and Granisetron. Typicalexamples of antibiotics that can be entrapped within the polymeric shellinclude Metronidazole, and Fusidic acid. Typical examples ofimmunomodulators that can be entrapped within the polymeric shellinclude Cyclosporine; and Biphenyl dimethyl dicarboxylic acid. Typicalexamples of anaesthetics that can be entrapped within the polymericshell include Propofol, Alfaxalone, and Hexobarbital.

With regard to anticancer agents in particular, the polymer of thepresent invention was found capable of entrapping completely within itspolymeric shell poorly-water soluble drugs or compounds like Paclitaxel,Docetaxel, Etoposide, and various Betulinic acid derivatives, such asthose designated as MJ-1098, DRF-4012 and DRF-4015 having the followingstructures (II), (III), and (IV), which in turn are disclosed in U.S.Pat. No. 6,403,816 and our pending Indian Application No. 265/DEL/2005,filed on Feb. 9, 2005.

The nanoparticulate pharmaceutical compositions of the poorlywater-soluble drugs or compounds mentioned hereinbefore and speciallythe nanoparticulate pharmaceutical compositions of the poorlywater-soluble drugs or compounds like Paclitaxel, Docetaxel andEtoposide and potent anticancer compounds like MJ-1098, DRF-4012 andDRF-4015, mentioned hereinbefore were found to have longer stability ofgreater than 24 hours in comparison to a nanoparticulate pharmaceuticalcomposition of Paclitaxel prepared as per the method of Burman et al,disclosed in U.S. Pat. No. 6,365,191, which as claimed in the saidpatent has a stability of only 12 hours. By reference to the term“stability”, it is to be construed to mean the period, counted in hours,wherein the poorly water-soluble drug or compound remains in solution inthe pharmaceutical composition comprising the same, without anyprecipitation of the drug or compound from therein.

Further, in the nanoparticulate pharmaceutical compositions of thepoorly water-soluble drugs or compounds, mentioned hereinbefore andspecially in the nanoparticulate pharmaceutical compositions of thepoorly water-soluble drugs or compounds like Paclitaxel, Docetaxel andEtoposide and potent anticancer compounds like MJ 1098, DRF 4012 and DRF4015, mentioned hereinbefore, the said drugs or compounds were found tobe entrapped within the polymeric micelle to an extent of greater than95% even after 24 hours. In comparison, in a nanoparticulatepharmaceutical composition of Paclitaxel prepared as per the method ofBurman et al, disclosed in U.S. Pat. No. 6,365,19, the drug was found tobe entrapped within the polymeric micelle to an extent of only 90% evenafter 24 hours.

A comparison of the pharmaceutical compositions of poorly water-solubledrugs or compounds in nanoparticulate form, comprising the polymer ofhigh purity and substantially free of monomeric contaminants of thepresent invention over the pharmaceutical composition of Paclitaxel,also in nanoparticulate form as disclosed in U.S. Pat. No. 6,365,191,would be evident from the comparison summarized in Table-IV.

TABLE IV Comparison Of The Pharmaceutical Composition Of PoorlyWater-Soluble Drugs Or Compounds Of The Present Invention Vis-à-vis ThePharmaceutical Composition Disclosed In U.S. Pat. No. 6,365,191 Sr.Disclosure Contained In No. Details U.S. Pat. No. 6,365,191 PresentInvention 1 Poorly water-soluble Drugs Taxane Derivatives, specially Ahost of Drugs and or Compounds referred to Paclitaxel Compounds 2 ThePolymer utilized in the Comprising of NIPAM, VP, and Comprising ofNIPAM, VP, Pharmaceutical Composition MPEG Monomeric Units and MPEGMonomeric Units 3 Monomeric Contaminants NIPAM: 0.066-0.076% w/w NIPAM:<0.001% w/w present in the Polymer VP: 0.008-0.011% w/w VP: <0.001% w/w4 Size of the Nanoparticles in Not Specified 30-150 nm thePharmaceutical Composition 4 Stability About 12 Hours >24 Hours 5Entrapment of the Drug 90% >95% within the Polymeric Micelle after 24Hours

In addition to the advantages of not only the polymer of high purity andsubstantially free of monomeric contaminants obtained as per the methodof the present invention but also the advantages the pharmaceuticalcompositions of poorly water-soluble drugs or compounds, comprising thepolymer of the present invention have over the polymers andpharmaceutical compositions of the prior art, as discussed in detailhereinbefore, the present inventors have further found a highlyselective method for preparation of pharmaceutical compositions ofpoorly water-soluble drugs or compounds, wherein the said poorlywater-soluble drug or compound, entrapped within the polymeric shell ofthe polymer utilized therein is produced in a nanoparticulate form ofconsistent size. This highly selective method forms another inventiveaspect of the present invention.

In the first place, it might be noted that from the descriptioncontained in Examples 1-40 and that given under the heading “ThePreferred Composition” and “Formulation for Infusion” in U.S. Pat. No.6,365,191 of Burman et al, the pharmaceutical composition of Paclitaxelis apparently prepared first by dissolving the requisite amount of thepolymer in a requisite amount of a diluting fluid (generally a 5% or 10%Dextrose solution), followed by addition of an anionic surfactant toobtain a clear solution, the pH of which is optionally adjusted with abuffering agent. To the resultant clear solution of the polymer and theanionic surfactant in the diluting fluid is then added an alcoholicsolution of Paclitaxel to obtain varying drug concentration ranging from0.1 to 10 mg/ml, which essentially is claimed to be the pharmaceuticalcomposition in nanoparticulate form useful for administration.

As mentioned hereinbefore, while U.S. Pat. No. 6,365,191 is silent aboutthe size of the nanoparticles thus obtained, it, however, was found toproduce or result in inconsistent sizes of the nanoparticles in thehands of the present inventors in their attempts to prepare apharmaceutical composition of Paclitaxel, as per the method described inExamples 1-40 and that given under the heading “The PreferredComposition” and “Formulation for Infusion” in U.S. Pat. No. 6,365,191of Burman et al.

Against this background, the present inventors found that production ofnanoparticles in consistent sizes was highly selective and dependslargely on:

-   i) The rate at which an alcoholic solution of Paclitaxel is added to    a solution of the polymer and other excipients in the diluting    fluid;-   ii) The volume of the alcoholic solution of Paclitaxel and the    volume of the diluting fluid, to which the former is added;-   iii) The internal diameter or bore size of the needle through which    the alcoholic solution of Paclitaxel is added to a solution of the    polymer and pharmaceutically acceptable excipients in the diluting    fluid; and-   iv) The position of the container, which contains the diluting fluid    and the polymer and pharmaceutically acceptable excipients at the    time of addition of the alcoholic solution of Paclitaxel.

In particular, the present inventors have found that only through:

-   a) Addition of an alcoholic solution of Paclitaxel through a syringe    to a solution of the polymer and other excipients in the diluting    fluid within a specified period of time;-   b) Utilization of a needle having an internal diameter of between    0.305 mm to 0.356 mm, for addition of a smaller volume of the    alcoholic solution of Paclitaxel to the solution of the polymer and    pharmaceutically acceptable excipients in the diluting fluid; or-   c) Utilization of a needle having an internal diameter of between    0.559 to 0.711 mm, for addition of a larger volume of the alcoholic    solution of Paclitaxel to the solution of the polymer and    pharmaceutically acceptable excipients in the diluting fluid;-   d) Injection of an alcoholic solution of Paclitaxel to the solution    of the polymer and other excipients in the diluting fluid, wherein    the needle of the syringe through which the alcoholic solution of    Paclitaxel is added shall remain dipped in the diluting fluid    solution; and-   e) Optionally, keeping the container containing the said diluting    fluid in an inverted position during injection of an alcoholic    solution of Paclitaxel to the solution of the polymer and other    excipients in the diluting fluid,    -   production of nanoparticles of consistent particle sizes, with        minimal or negligible size variation and consistent loading of        the drug, within the polymeric shell could be achieved. Further,        only through the selective method it was found a longer        stability of the pharmaceutical composition could be achieved.

It was further found that the highly selective method, mentionedhereinbefore is not limited to production of nanoparticles of Paclitaxelonly, but also is equally effective in production of nanoparticles ofconsistent particle sizes of other poorly water-soluble drugs orcompounds, especially Docetaxel, Etoposide, Betulinic acid, potentanticancer betulinic acid derivatives like MJ-1098 of formula (II),DRF-4012 of formula (III), DRF-4015 of formula (IV), referred tohereinbefore.

It was particularly found that if the addition time of a smaller volume,say of between 1 to 5 ml of the solution of the poorly water-solubledrug or compound to a solution (about 35 times the volume injected) ofthe polymer and other excipients in the diluting fluid, exceeds a timeof 4 seconds, or if the internal diameter of the needle (through whichthe solution of the poorly water-soluble drug or compound is injected)is outside the range of 0.305 mm to 0.356 mm, then such an additionresults in production of inconsistent particle sizes of thenanoparticles of the poorly water-soluble drugs or compounds as well asresults in a pharmaceutical composition possessing poor stability,meaning whereby the solution does not remain clear for longer periods oftime but becomes opalescent in shorter periods of time.

Similarly, it was particularly found that if the addition time of alarger volume, say of between 5 to 15 ml of the solution of the poorlywater-soluble drug or compound to a solution (about 35 times the volumeinjected) of the polymer and other excipients in the diluting fluid,exceeds a time of 10 seconds, or if the internal diameter of the needle(through which the solution of the poorly water-soluble drug or compoundis injected) is outside the range of 0.559 to 0.711 mm, then such anaddition results in production of inconsistent particle sizes of thenanoparticles of the poorly water-soluble drugs or compounds as well asresults in a pharmaceutical composition possessing poor stability,meaning whereby the solution does not remain clear for longer periods oftime but becomes opalescent in shorter periods of time.

This could be exemplified with respect to pharmaceutical compositions oftwo poorly water-soluble anticancer drugs or compounds, viz. Paclitaxeland a Betulinic acid derivative, DRF-4012 of formula (III), wherein theeffect of the time of addition of a solution of the said drugs and theinternal diameters of the needles through which such solutions are addedto the solution of the polymer and pharmaceutically acceptableexcipients in the diluting fluid are summarized in Table-V.

The pharmaceutical composition of the present invention is convenientlypresented as a two vial kit:

-   a) one comprising a solution of a poorly water-soluble drug or    compound in a water-miscible solvent, or mixtures thereof at a    suitable concentration of the said drug; and-   b) the other comprising a solution of the polymer of the present    invention, of high purity and substantially free of monomeric    contaminants, along with pharmaceutically acceptable excipients in    an aqueous solvent, generally water of injection grade, both the    vials being sterile and manufactured and packed under aseptic    conditions.

The contents of the two vials are then added to the diluting fluid priorto administration to humans/animals.

Optionally, the kit can further comprise a diluting fluid, and a syringeand a needle having an internal diameter in the range of 0.305 to 0.356mm, if a small volume, say 1-5 ml of the contents of vial a) are to beadded to about 35 times its volume of the contents of vial b) or asyringe and a needle having an internal diameter in the range of 0.559to 0.711 mm, if a larger volume, say 10-15 ml of the contents of vial a)are to be added to about 35 times its volume of the contents of vial b).

Especially in the case of a kit comprising Paclitaxel, foradministration to patients for treatment of breast cancer, the two vialkit presentation would comprise of:

-   a) one vial containing a solution of 400 mg of Paclitaxel in 20 ml    of ethanol;-   b) the other containing a solution containing 200 mg of the polymer    of the present invention, of high purity and substantially free of    monomeric contaminants, along with pharmaceutically acceptable    excipients in 20 ml of water, and    -   optionally, the kit can further comprise a 500 ml bottle of 10%        Dextrose solution, and a syringe and a needle having an internal        diameter of 0.711 mm, for injection of the solution of vial a)        to the 500 ml bottle of 10% Dextrose solution, to which has been        added the solution of vial b) in a time period not exceeding 10        seconds, preferably in a time period of 6 to seconds to produce        a pharmaceutical composition suitable for administration in        nanoparticles of consistent particle sizes, with minimal or        negligible size variation and consistent loading of the drug,        within the polymeric shell, and longer stability.

TABLE V Effect Of The Time Of Addition Of A Solution Of PoorlyWater-Soluble Drugs Or Compounds And Internal Diameter Of The NeedlesThrough Which Such Solutions Are Added To A Solution Of The Polymer AndOther Excipients In A Diluting Fluid Internal Diameter (in Addition Timemm) Of The Needle (In Secs). Of The Through Which The Solution OfSolution Of The Poorly Water- Poorly Water-Soluble Soluble Drug OrPoorly Water-Soluble Drug Or Compound Compound To Drug Or Compound; TheIs Added To The The Solution Of Solvent In Which It Is Solution Of TheThe Polymer And Observations/Results Dissolved; And The Polymer AndExcipients In A Average Clarity Sr. Volume Of Solution Excipients In ADiluting Fluid Particle Stability Of The No. Injected Diluting Fluid (Inml) Size (nm) (Hrs) Solution 1 Paclitaxel/Ethanol (1 ml) 0.305 3Secs/≈35 ml 80 >24 Clear 2 Paclitaxel/Ethanol (1 ml) 0.467 2 Secs/≈35 ml90 20 Very Slight Opalescence 3 Paclitaxel/Ethanol (1 ml) 0.711 2Secs/≈35 ml 135 <10 Slight Opalescence 4 Paclitaxel/Ethanol (1 ml) 1.2702 Secs/≈35 ml 270 <4 Opalescent 5 Paclitaxel/Ethanol (1 ml) 0.305 6Secs/≈35 ml 240 <4 Slight Opalescence 6 Paclitaxel/Ethanol (1 ml) 0.4676 Secs/≈35 ml 280 <2 Highly Opalescent 7 Paclitaxel/Ethanol (1 ml) 0.7116 Secs/≈35 ml 285 <2 Highly Opalescent 8 Paclitaxel/Ethanol (1 ml) 1.2706 Secs/≈35 ml 2300 <0.5 Milky 9 DRF-4012 of Formula 0.305 3 Secs/≈35 ml70 >24 Clear (III)/Ethanol (1 ml) 10 DRF-4012 of Formula 0.465 2Secs/≈35 ml 90 20 Very Slight (III)/Ethanol (1 ml) Opalescence 11DRF-4012 of Formula 0.711 2 Secs/≈35 ml 100 <10 Slight (III)/Ethanol (1ml) Opalescence 12 DRF-4012 of Formula 1.270 2 Secs/≈35 ml 130 <6Opalescent (III)/Ethanol (1 ml) 13 DRF-4012 of Formula 0.305 6 Secs/≈35ml 200 <6 Slight (III)/Ethanol (1 ml) Opalescence 14 DRF-4012 of Formula0.467 6 Secs/≈35 ml 200 <4 Highly (III)/Ethanol (1 ml) Opalescent 15DRF-4012 of Formula 0.711 6 Secs/≈35 ml 240 <2 Highly (III)/Ethanol (1ml) Opalescent 16 DRF-4012 of Formula 1.270 6 Secs/≈35 ml 8100 <0.5Milky (III)/Ethanol (1 ml) 17 Paclitaxel/Ethanol (15 ml) 0.711 10Secs/≈500 ml 85 >24 Clear 18 Paclitaxel/Ethanol (15 ml) 0.711 15Secs/≈500 ml 150 <15 Slight Opalescence 19 Paclitaxel/Ethanol (15 ml)0.330 18 Secs/≈500 ml 50 20 Clear

In summary, the present invention, as mentioned hereinbefore, is a stepforward in providing a solution to most, if not all of the limitationsof the prior art methods in the field of nanoparticle technology andprovides:

-   i) A polymer comprising three monomeric units selected from NIPAM,    VP, and MPEG, of high purity and substantially free of monomeric    contaminants, with the level of toxic NIPAM and VP in the    polymer<0.001%; which, moreover, has been established to be    biocompatible, non-biodegradable, safe and non-toxic for    human/animal use;-   ii) A highly selective method for preparation of the polymer    comprising three monomeric units selected from NIPAM, VP, and MPEG,    of high purity and substantially free of monomeric contaminants,    with the level of toxic NIPAM and VP in the polymer<0.001%    comprising subjecting an aqueous solution of the polymer thus    prepared to diafiltration;-   iii) A pharmaceutical composition of poorly water-soluble drugs or    compounds in nanoparticulate form, comprising the polymer of high    purity and substantially free of monomeric contaminants along with    pharmaceutically acceptable excipients, which is safe and non-toxic    and hence highly suitable for human/animal use or administration;-   iv) A highly selective method for production of a pharmaceutical    composition of poorly water-soluble drugs or compounds in    nanoparticulate form, comprising the polymer of high purity and    substantially free of monomeric contaminants along with    pharmaceutically acceptable excipients, having consistent particle    sizes of the nanoparticles and consistent drug loading; and-   v) A pharmaceutical composition of poorly water-soluble drugs or    compounds in nanoparticulate form, comprising the polymer of high    purity and substantially free of monomeric contaminants along with    pharmaceutically acceptable excipients, having consistent particle    sizes of the nanoparticles with higher drug loading and a longer    stability.

DETAILED DESCRIPTION OF THE INVENTION A. Preparation of the Polymer ofthe Present Invention

The polymer of the present invention comprises of the three monomericunits selected from NIPAM, VP and MPEG, wherein the polymer chains arecross-linked with a cross linking agent, which does not contain anysulfhydryl groups or disulfide bonds.

The cross-linking agent plays an important role during polymerization byproviding cross-links into the linear polymer chains and is in general abi-functional vinyl derivative, whenever used. It can be more thanbi-functional i.e. it can have more than two reactive sites. Abi-functional vinyl derivative that can be advantageously employed isN,N′-Methylene bis acrylamide (MBA), which is preferred.

The polymer of the present invention can be prepared by general methodsnormally adopted for polymerization reactions.

In a particular embodiment, the polymer of the present invention can beprepared by subjecting the monomers N-Isopropylacrylamide (NIPAM),1-Vinyl-2-pyrrolidone (VP) and Polyethylene glycol (mol. wt 6000) esterof Maleic anhydride (MPEG) for free radical polymerization in presenceof an activator, a polymerization initiator, and a cross-linking agentin aqueous medium.

A combination of monomers, N-isopropyl acrylamide (NIPAM) andVinylpyrrolidone (VP) could be employed in the weight ratio rangingbetween 55:22 to 65:35, while the comonomeric composition of (NIPAM+VP):MPEG that can be employed is in the range of 80:20 to 95:5. Moreparticularly and preferably, a combination of monomers-N-isopropylacrylamide (NIPAM) and Vinylpyrrolidone (VP) is employed in the weightratio ranging between 58:32 to 62:28 and the comonomeric composition of(NIPAM+VP): MPEG is employed in the range of 90:10 or 95:5, which isfound to impart the desired biocompatibility, non-biodegradability andbiologically safe profile to the polymers, for the reason that thisparticular ratio consistently results in formation of a randomlyhyperbranched co-polymeric unit of NIPAM and VP, which are stabilized byan outer shell coating formed from hydrogen-bonding by the diesteradduct (major) and monoester adduct (minor) of maleicanhydride-polyethylene Glycol (MPEG).

The polymerization initiators play an important role in initiation offree radical formation. The initiators that can be employed can beperoxide compounds, such as diacyl peroxide, benzoyl peroxide, diacetylperoxide, dialkyl peroxides, tertiary butyl peroxide and tertiary amylperoxide or nitrile based polymerization initiators such as 2,2′-Azobisisobutyronitrile (AIBN) or inorganic salt based polymerizationinitiators such as Ammonium perdisulphate or Ammonium persulphate (APS),used either alone or in combination.

Amongst the abovementioned polymerization initiators, Ammoniumpersulphate (APS) is the preferred one.

Although, polymerization initiators initiate the polymerization,however, the polymerization reaction is found to be accelerated by thepresence of activating agents (often known as Activators) which catalyzethe formation of free radicals from polymerization initiators. Suchactivators may be selected from Tetramethylethylene diamine (TMED) andFerrous Ammonium Sulphate (FAS), of which a combination of TMED and FASis preferred. Any combination of the polymerization initiator and theactivator can be employed for the polymerization reaction. Two or moreinitiators can also be used. Similarly, two or more activators can alsobe employed.

As mentioned hereinbefore, the cross-linking agent plays an importantrole during polymerization by providing cross-links into the linearpolymer chains and is in general a bi-functional vinyl derivative,whenever used. It can be more than bi-functional i.e. it can have morethan two reactive sites. A bi-functional vinyl derivative that can beused is N,N′-methylene bis acrylamide (MBA), which is preferred.

The polymerization is carried out in the presence of an inert gas, whichcan be nitrogen or argon.

Generally, the polymerization reaction is carried out, first bydissolving an appropriate quantities of the respective monomers viz,N-Isopropylacrylamide (NIPAM), 1-Vinyl-2-pyrrolidone (VP) andPolyethylene glycol (mol. wt 6000) ester of Maleic anhydride (MPEG) inan aqueous solvent, which in general is water. To the aqueous solutionof the respective monomers thus obtained is added in succession anaqueous solution of a cross-linking agent and an activator. The solutionis de-aerated by bubbling an inert gas for about 30-60 minutes. To thede-aerated solution is added an aqueous solution of polymerizationinitiators and the solution is subjected to polymerization at atemperature of between 25° to 45° C., preferably at a temperaturebetween 25° to 35° C. under continuous inert gas bubbling for a periodof time till the polymerization is complete.

The cross-linking agent can be employed in quantities in the range ofbetween 1.3-1.5% w/w of the total monomer content, and more preferablyin the range of between 1.35-1.4% w/w of the total monomer content.

Activators can be employed in quantities in the range between 15-18% w/wof the total monomer content and more preferably in the range between15-16% w/w of the total monomer content.

The polymerization initiator can be employed in quantities in the rangeof between 20-30% w/w of the total monomer content and more preferablyin the range of between 23-25% w/w of the total monomer content.

The progress of the polymerization reaction is monitored by HPLC andusually gets completed in about 3-6 hours.

After completion of the polymerization reaction, the solution issubjected to filtration through pre-sterilized, disposable 0.2 μmPolyethersulphone membrane 1″ capsule filters, 0.8 and 0.2 μm pore size;Type DPS-5101AA-201 (Make: M/s Advanced Microdevices Pvt. Ltd, India).The filtered contents of the reaction vessel are subjected toDiafiltration using Proflux M12 (Millipore) diafiltration device toremove monomeric contaminants and other low molecular weight impurities.

The diafiltration is generally completed in less than one hour andnormally results in not only a solution substantially free of monomericcontaminants but also in a concentrated form, usually one-forth toone-sixth (¼^(th) to ⅙^(th)) of the initial volume of the solutionsubjected to diafiltration. If necessary, the concentrated solution,thus obtained, which is substantially free of monomeric contaminants,can be subjected to another cycle of diafiltration. The concentratedsolution of polymer of the present invention of high purity andsubstantially free of monomeric contaminants can be subjected to a stepof lyophilization to obtain the polymer in solid lyophilized form forutilization in pharmaceutical compositions or the concentrated solutionas such can be directly utilized for formulation of the saidpharmaceutical compositions.

In a typical embodiment, the polymerization reaction is carried out, bydissolving an appropriate quantity of the respective monomers viz,N-Isopropylacrylamide (NIPAM), 1-Vinyl-2-pyrrolidone (VP) andPolyethylene glycol (mol. wt 6000) ester of Maleic anhydride (MPEG) inwater. To the aqueous solution of the respective monomers thus obtained,is added an appropriate volume of aqueous solution (about 5% w/v) of across-linking agent, N,N′-methylene bis acrylamide (about 1.37% w/w ofthe total monomer content) and a combination of activators, comprisingan appropriate volume of Tetramethylethylene diamine (TMED, about 15.4%w/w of the total monomer content) and an aqueous solution (0.5% w/v) ofFerrous Ammonium Sulphate (about 0.1% w/w of the total monomer content).It is preferable to add one of the activators first and the other onealong with the polymerization initiator, which is added later. Thesolution is de-aerated by bubbling nitrogen for about 30 minutes. To thede-aerated solution is added an appropriate volume of an aqueoussolution (about 80% w/v) of polymerization initiators, Ammoniumpersulphate (about 24% w/w of the total monomer content) and thesolution is subjected to polymerization at a temperature preferablybetween 25° to 35° C. under continuous nitrogen bubbling for a period oftime till the polymerization is complete. Usually, the polymerizationreaction gets completed in 3-5 hours.

After completion of the polymerization reaction, the solution issubjected to filtration through pre-sterilized, disposable 0.2 μmPolyethersulphone membrane 1″ capsule filter (0.8+0.2 μm pore size). Thefiltered contents of the reaction vessel are subjected to diafiltrationto remove monomeric contaminants and other low molecular weightimpurities.

In a typical embodiment, a solution of the polymer in water at aconcentration of say 1 gm in 50 ml could be subjected to diafiltration,whereupon, after the diafiltration a concentrated solution of thepolymer in about one-fourth to one-sixth (¼^(th) to ⅙^(th)) of theinitial volume, say 1 gm of the polymer in about 12-13 ml of water isobtained, which contains less than 0.001% w/w of both NIPAM and VP.

The detection and quantification of the residual monomers, especiallyresidual VP and NIPAM in the polymer were carried out by HPLC. The HPLCsystem that can be used for detection of the monomers is Agilent 1100series or equivalents, using Reverse Phase RP-18 (C-18) columns[Lichrospher RP-18e, 5μ, 250 mm×4 mm]. The Mobile Phase used is amixture of water and acetonitrile in a ratio of 80:20, at a flow rate of1 ml/min, with a sample injection volume is 50 μl.

The run time is 10 mins and the column temperature is 30° C. and theDetector wavelength is 226 nm.

Under the above conditions, NIPAM had a retention time of about 3minutes, whereas VP had a retention time of about 5 minutes.

The concentrated solution of polymer so obtained is of high purity andsubstantially free of monomeric contaminants, which can be subjected toa step of lyophilization to obtain the polymer in solid lyophilized formfor utilization in pharmaceutical compositions or the concentratedsolution as such can be directly utilized for formulation of the saidpharmaceutical composition. It is however preferable to utilize theconcentrated solution of the polymer as such for formulation intopharmaceutical compositions.

B. Characterization of the Polymer of the Present Invention

The polymer of the present invention obtained by the method mentionedhereinbefore was subjected to extensive spectroscopic analysis such¹H-NMR, ¹³C-NMR, Fourier Transform Infrared (FT-IR) and Thermal analysissuch as Differential Scanning Calorimetry (DSC) and Thermo Gravimetricanalysis (TGA) etc. to elucidate the structure of the polymer thusobtained.

The ¹H NMR spectrum of the polymer of the present invention in CDCl₃shows peaks at the δ (ppm) of 1.14 (br, —CH(CH₃)₂); 1.45 (br,—CH₂—CH—N(VP-Ring); 1.63 (br, —CH₂—CHC(═O)NH); 1.99 (br, —CH C(═O)NH—),CH₂ (VP ring), 2.36 (CH₂, VP ring), 3.0 (—O—CH₂—CH₂—), 3.23 (CH₂—N—);3.62-3.66 (Br, CH₂, MPEG); 3.72 (NH—CH(CH₃)₂); 3.97 (Br, CH). The ¹H-NMRspectrum of the polymer of the present invention is summarized in FIG.1.

The ¹³C NMR spectrum of the polymer of the present invention shows peaksat the δ (ppm) of 174 (C═O); 76.6-77.6 (multiplet for CDCl₃ and CH forpolymer backbone); 70.6 (CH₂'s MPEG); 41.6 (CH for isopropyl unit); 31.8(CH₂ 's, polymer backbone); 22.6 (CH₃ 's, isopropyl). The ¹³C-NMRspectrum of the polymer of the present invention is summarized in FIG.2.

The Fourier Transform Infrared (FT-IR) spectrum of the polymer of thepresent invention shows peaks at the following frequency values (cm⁻¹)of 3500 (s, OH); 3296 (s, NH, sec-Amide); 2972-2933 (s, CH, CH₂, CH₃);1651 (br, strong, split peaks ester C═O and C═O of amide I); 1546 (s, NHbend of Amide II and possibly C═O of free acid, minor); 1387, 1367(doublet of Isopropyl groups, CH₃, deformation); 1172-1129 (m, O—C—O).The Fourier Transform Infrared (FT-IR) spectrum of the polymer of thepresent invention is summarized in FIG. 3.

These characterization studies confirm that the polymer of the presentinvention has the structure, which is depicted in hereinbelow as formula(I):

Further, in order to characterize the physicochemical properties of thepolymer in detail, various properties of the polymer such as thermalproperties, Critical Micelle Concentration (CMC), solubility and pH,storage stability were evaluated.

Thermogravimetric analysis (TGA), showed that there is some weight lossfrom 51-260° C., which indicates loss of solvent and some macromolecularreactions that might be occurring especially in the MPEG units of thepolymer before degradation starts occurring at around 310° C. Thisindicates that the polymer has high thermal stability, which in greatpart may be provided by the MPEG units. The TGA thermogram of thepolymer of the present invention is summarized in FIG. 4.

Further, the Differential Scanning Calorimetry (DSC) profile of thepolymer, represented in FIG. 5 did not show any glass transitiontemperature (Tg), but only a melting temperature (Tm) of 58° C. and arecrystallization point temperature (Tc) of 38.4° C. were observed. Theabsence of any clear Tg may be indicative of a highly rigidhyperbranched structure, which could also be contributed by extensivehydrogen bonding with MPEG.

Structure of the Polymer of the Present Invention

Lower Critical Solution Temperature (LCST) of the polymer has values inthe region of 50-60° C. Again this is an important parameter foramphiphilic polymers in aqueous phase manifesting in thermo-responsivephase transitions at a certain temperature called LCST. Below the LCSTthe polymer would exhibit a soluble extended chain configuration i.e.hydrophilic behaviour. Above the LCST, the polymer undergoes a phasetransition to conform to forming an insoluble, hydrophobic aggregate.This property is useful to determine the ability to form micelles in theappropriate solvent and act in delivery systems for the drugs inpharmaceutical applications.

Critical Micelle Concentration (CMC) is another important parameter thatdefines the encapsulating ability of a nano-carrier and determines thestability. This is the lowest concentration for the amphiphilic polymeror unimers to form a micellar structure capable of encapsulating a drugin its hydrophobic core. The CMC value for present polymer is about 0.2mg/ml. Further, the polymer comprising of thermo-sensitive and pHsensitive monomers such as N-isopropylacrylamide (NIPAM) and 1-VinylPyrrolidin-2-one (VP) are well known as biocompatible with proteins andblood cells. Further, biomedical applications of poly (NIPAM) are quitewidespread due to its reversible temperature transition i.e. LCST,excellent Hydrogen-bonding, micellar and hydrogel forming capabilities.Similarly, Poly Vinylpyrrolidone (also known as Povidone) polymers arealso highly water soluble and form extensive Hydrogen-bonding withwater. The intended application of this polymer was to design a novelincorporating system incorporating the strengths of the variouspre-defined monomers leading to the formation of a thermo-sensitive, pHsensitive, stable polymeric nanoparticles containing hydrophilic andhydrophobic groups to solubilize the drugs that are poorly soluble inwater.

Very surprisingly, it was found that the formation of a randomlyhyperbranched co-polymeric unit consisting of NIPAM and VP stabilized byan outer shell coating formed from hydrogen-bonding by diester adduct(major) and monoester adduct (minor) of maleic anhydride-polyethyleneGlycol (MPEG) having the comonomeric composition of (NIPAM+VP): MPEG inthe range of 80:20 to 95:5 as well as NIPAM: VP units in the range of55:22 to 65:35 imparts the desired biocompatibility,non-biodegradability and biologically safe profile to polymers.Specifically, it was found that better results (higher LCST, higheryield, percentage release from Paclitaxel nanoparticles) were obtainedwhen the composition of (NIPAM+VP): MPEG is in the range of 90:10 or95:5 and NIPAM: VP units are used in the range of 58:32 to 62:28. Theratio of the monomer used is also consistent in the final polymer and isconfirmed by various studies such as ¹H-NMR, ¹³C-NMR and FourierTransform Infrared (FT-IR) spectral studies

C. Biocompatibility and Non-Biodegradability of the Polymer of thePresent Invention

When Pharmacokinetics, Biodistribution and Elimination of [¹⁴C]-labelledpolymer was evaluated using male swiss albino mice, the radioactiveblood concentration profile revealed a bi-phasic curve (FIG. 6), withshort elimination half-life T_(1/2)(β) of 0.448±0.157 hours (26.88 min)and rapid clearance of 54.7 ml/hr. The results of this study aresummarized in Tables-VI and VII.

TABLE VI Pharmacokinetic Parameters Of The Polymer Of The PresentInvention Parameter Estimate ± SE T_(1/2)(K10) 0.152 ± 0.018 hrT_(1/2)(alpha) 0.065 ± 0.014 hr T_(1/2)(beta) 0.448 ± 0.157 hr C_(max)82.96 ± 5.11 μg/mL AUC 18.29 ± 1.62 hr × μg/ml CL 54.67 ± 4.86 ml/hr MRT0.465 ± 0.13 hr V_(ss) 25.43 ± 5.2 ml

The dominant route of elimination was found to be urine (urine, 66.91%vs feaces, 17.39% at 48 hrs) and recovery data collected up to 48 hrsaccounts for 84.87% of radioactivity injected. Tissue distribution wasnegligible. The kidney, liver, skin and intestine were found to be thetarget organs. However, the level of the polymer in tissues was rapidlycleared via urine and faeces.

TABLE VII Recovery of Radio-labelled Polymer Of The Present InventionPercentage (%) of dose Time 0-10 min 0-1 hr 0-24 hrs 0-48 hrs Urine27.14 61.64 64.56 66.91 Feaces 0.10 0.65 12.29 17.39 Tissues 15.50 3.220.78 0.57 Rinse 5.04 2.27 0.84 0.00 Total 47.16 67.78 78.47 84.87

Thus, in conclusion, the polymer is found to be rapidly eliminated fromthe body without being deposited and degraded in vital organs suggestingthe safety and utility of the polymer for human use.

D. Toxicity Studies on the Polymer of the Present Invention

Toxicity studies of Polymer of formula (I) were carried out to evaluate:

(i) Localised Toxicity (Subcutaneous and Intravenous);

(ii) Target Organs Dose Toxicity up to 800 mg/kg animal body weight; and(iii) Six Months Cyclical dose toxicity

D(i) Localised Toxicity (Subcutaneous and Intravenous)

The toxicity of the polymer was determined after a single subcutaneousadministration of 100 μl of 75-mg/ml of the polymer in the rabbit ear,which caused mild inflammation at the site of injection, when testedafter 48 hrs post injection, suggesting that the present polymer doesnot cause any local toxicity at the site of administration followingsubcutaneous administration.

The toxicity of the present polymer was determined for a five daycontinuous intravenous administration of 75-mg/ml of present polymer ata dose of 125 mg/kg in rabbit ear vein and similar results wereobtained, further confirming that the present polymer does not cause anylocal toxicity at the site of administration.

Representative photographs of S&E stained Rabbit Ear Lobe site after 48hours of subcutaneous injection with 10% Dextrose solution is shown inFIG. 7 and photographs of S&E stained Rabbit Ear Lobe site after 48hours of subcutaneous injection with an aqueous solution of the polymeris shown in FIG. 8.

Representative photographs of S&E stained Rabbit's Marginal Ear Veinsite after 24 hours of intravenous injection with 10% Dextrose solutionis shown in FIG. 9 and photographs of S&E stained Rabbit's Marginal EarVein site after 24 hours of intravenous injection with an aqueoussolution of the polymer is shown in FIG. 10.

D(ii) Target Organs Dose Toxicity (Up to 800 mg/kg Animal Body Weight)

Further, the toxicity was evaluated on possible target organ(s) withspecial reference to microvasculature and determined by singleintravenous bolus administration in Wistar Rats. The polymer wasadministered at two different dosages, viz. 400 mg/kg and 800 mg/kg.Under the conditions of study, single intravenous bolus administrationof the present polymer at any dose does not produce any mortality or anyobservable toxic sign or symptoms in rats. Individual and Mean bodyweights of rats showed a steady increase in both Polymer treated andcontrol groups. No significant difference was noted for body weight fortreated animals at both doses as compared to that of the control.

In rats treated with the polymer, haematological parameters were withinnormal limits throughout the study. Biochemical parameters were alsowithin the normal limits for the animals treated with both doses. Thephotoactometer test showed that there was no significant differencebetween the locomotor activity between the Control and Treated groups onday 7 and 21 respectively suggesting that the polymer does not have anyneurotoxicity.

The Treated and Control group specimens showed similar histologicalfeatures. Histology study was performed on vital organs such as liver,heart, lungs, kidneys, spleen, stomach, colon, thigh muscle and eye. Allorgans studied showed normal structure on light microscopic examination.The microvasculature in each organ was carefully examined and nopathological features were seen in any of the organs. Further, therewere no changes in microvasculature of the polymer treated animals.

From the above observations, it was abundantly evident that polymer ofthe present invention at a dose of either 400 mg/kg or 800 mg/kg of bodyweight administered for five consecutive days did not cause any generaltoxicity or any significant haematological toxicity indicating thebiologically safe and non-toxic profile of the present polymer.

D(iii) Six Months Cyclical Dose Toxicity

Further, six months cyclical dose toxicity was studied in rats byintravenous injection of polymer used in nanoparticle formulation.Male/Female Wistar rats were used for the study and dosing was doneintravenously in the lateral tail vein cyclically once every three weeksfor a period of 180 days (approximately 26 weeks). Animals of treatedand control groups remained generally active and healthy during theperiod of study. The polymer concentration equivalent to 10 mg/kg ofdrug was found to be safe in the animals under study. Minimumalterations in haematology parameters noticed were within the normalrange for Wistar rats and were not found to be treatment related.

The above studies suggest that the synthesised polymer is non-toxic andbiologically safe for use in making pharmaceutical compositions.

E. Pharmaceutical Compositions Comprising the Polymer of the PresentInvention

As discussed hereinbefore, the polymer of the present invention offormula (I), of high purity and substantially free of monomericcontaminants, in particular having residual monomeric NIPAM andVP<0.001% could be utilized to advantage for preparation ofpharmaceutical compositions of poorly water-soluble drugs or compoundsin nanoparticulate form, which are safe and non-toxic for human/animaladministration or use.

In particular, the polymer of the present invention could be used toprepare a pharmaceutical composition in nanoparticulate forms, alongwith pharmaceutically acceptable excipients entrapping a host of poorlywater-soluble drugs or compounds completely or near-completely withinits polymeric shell.

Further, as discussed hereinbefore, poorly water-soluble drugs orcompounds that can be utilized in the pharmaceutical compositions of thepresent invention are those generally having water solubility of lessthan 10 mg/ml.

Examples of such poorly water-soluble drugs or compounds include, butare not limited to, anticancer agents, anti-inflammatory agents,anti-fungal agents, antiemetics, antihypertensive agents, sex hormones,steroids, antibiotics, immunomodulators, anaesthetics etc. Typicalexamples of anticancer agents that can be entrapped within the polymericshell are Paclitaxel, Docetaxel, and other related taxane derivatives;Irinotecan, Topotecan, and other related Camptothecin derivatives;Doxorubicin, Daunomycin, and related Anthracycline derivatives;Cisplatin; Oxaliplatin; 5-Fluorouracil; Mitomycin; Methotrexate;Etoposide; Betulinic acid and its derivatives; and Wedelolactone and itsderivatives. Typical examples of anti-inflammatory agents that can beentrapped within the polymeric shell include Indomethacin, Ibuprofen,Ketoprofen, Flubiprofen, Piroxicam, Tenoxicam, and Naproxen. Typicalexamples of anti-fungal agents that can be entrapped within thepolymeric shell include Ketoconazole, and Amphotericin B. Typicalexamples of sex hormones that can be entrapped within the polymericshell include Testosterone, Estrogen, Progesterone, and Estradiol.Typical examples of steroids that can be entrapped within the polymericshell include Dexamethasone, Prednisolone, and Triamcinolone. Typicalexamples of antihypertensive agents that can be entrapped within thepolymeric shell include Captopril, Ramipril, Terazosin, Minoxidil, andParazosin. Typical examples of antiemetics that can be entrapped withinthe polymeric shell include Ondansetron and Granisetron. Typicalexamples of antibiotics that can be entrapped within the polymeric shellinclude Metronidazole, and Fusidic acid. Typical examples ofimmunomodulators that can be entrapped within the polymeric shellinclude Cyclosporine; and Biphenyl dimethyl dicarboxylic acid. Typicalexamples of anaesthetics that can be entrapped within the polymericshell include Propopol, Alfaxalone, and Hexobarbital

A pharmaceutical composition of poorly water-soluble drugs or compoundstypically comprises of a two kit-vial presentation, comprising in onehand, a vial containing a solution of a poorly water-soluble drug in awater-miscible solvent, or mixtures thereof, at a suitable concentrationof the said drug or compound; and comprising on the other hand, a vialcontaining a solution of the polymer of the present invention, of highpurity and substantially free of monomeric contaminants andpharmaceutically acceptable excipients in an aqueous solvent, generallywater of injection grade, both the kit vials being sterile andmanufactured and packed under aseptic conditions. The contents of thetwo vials are then added in succession to a diluting fluid foradministration to humans/animals. It should be noted, as discussedhereinbefore as well as would be discussed hereinlater, the poorlywater-soluble drug or compound gets entrapped within the polymeric shellof the polymer utilized therein and is produced in a nanoparticulateform of consistent size. The ratio of the solution of a poorlywater-soluble drug in a water-miscible solvent, or mixtures thereof tothe a solution of a poorly water-soluble drug in a water-misciblesolvent, or mixtures thereof, contained in the two is generally between1:1 to 1:10 by volume, preferably in a ratio of 1:1.

Optionally, the two kit-vial presentation can further comprise adiluting fluid, and a syringe and a needle of internal diameter in therange of between 0.305 to 0.356 or 0.559 to 0.711 mm, which depends onthe volume of the drug solution and the volume of the diluting fluidcontaining the polymer and excipients to be mixed for administration tohumans/animals in need thereof.

Suitable water-miscible solvents that can be utilized for dissolving thepoorly water-soluble drug or compound include an aliphatic alcohol,specially ethanol; dialkyl amides, specially dimethyl formamide anddimethyl acetamide; dialklyl sufoxides, specially dimethyl sulfoxide anddiethyl sulfoxide; polyethylene glycols of various molecular weights;polypropylene glycols of various molecular weights; surfactants,specially polysorbate 80, polysorbate 20, polyoxyethylated vegetableoil, and polyethoxylated castor oil; glycerine etc.

The pharmaceutically acceptable excipients that can be used to advantageinclude sodium deoxycholate; various bile salts; polysorbates of variousgrades, specially polysorbate 80, polysorbate 20, polyoxyethylatedvegetable oil, and polyethoxylated castor oil; polysaccharides likedextrose, sucrose, lactose, mannitol etc.; sorbitan esters or spans ofvarious grades; myrj of various grades; poloxomers of various gradesetc., and a buffering agent for adjustment of the pH. Any bufferingagent known in the art can be employed for adjustment of the pH of thesolution, and in a preferred embodiment it is advantageous to utilizesodium citrate as the buffering agent.

Of the pharmaceutically acceptable excipients, sodium deoxycholate ispreferred since it has an effect in stabilization of the pharmaceuticalcomposition, whereas the buffering agent is used to adjust the pH of theperfusion fluid in the range of between 6.0 to 8.5, which is also foundto have an effect in stabilization of the pharmaceutical composition.

The pharmaceutical composition can have a suitable loading or dose ofthe poorly water-soluble drug or compound and selection of an optimumloading or dose of the said drug or compound, largely depends on thenature of the drug or compound, its solubility as well as to thetherapeutic disorder for which it is administered for. In the case ofthe pharmaceutically acceptable excipients, the proportion or quantityof that can be utilized in the pharmaceutical composition similarly, inturn depends on nature and loading of the poorly water-soluble drug orcompound contained in the composition.

The pharmaceutical composition in nanoparticulate of poorlywater-soluble drugs or compounds of the present invention can beprepared the following way:

-   i) Preparation of the drug concentrate, comprising dissolving the    poorly water-soluble drug or compound in a suitable water-miscible    solvent, or mixtures thereof;-   ii) Preparation of an aqueous concentrate of the polymer and    pharmaceutically acceptable excipients, comprising the steps of:    -   a) first addition of the requisite amount of the polymer of        formula (I), of high purity and substantially free of monomeric        contaminants, specially having a level of toxic NIPAM and        VP<0.001% to an appropriate quantity of water-for-injection to        obtain a solution;    -   b) addition of pharmaceutically acceptable excipients and a        buffering agent to the solution of the polymer in water;-   iii) Mixing the solution of step ii b) with a diluting fluid to get    a clear solution;-   iv) Utilization of a needle having an internal diameter of between    0.305 to 0.356 mm, for addition of a smaller volume of solution of    step i) to the solution of step iii); or-   v) Utilization of a needle having an internal diameter of between    0.559 to 0.711 mm, for addition of a larger volume of the solution    of step i) to the solution of step iii);-   vi) Injection of the solution of step i) to the solution of step    iii), wherein the needle of the syringe through which the solution    of step 1) is added shall remain dipped in the solution of step    iii); and-   vii) Optionally, keeping the container containing the solution of    step iii) in an inverted position during injection of the solution    of step i),    so as to completely entrap the poorly water-soluble drug or compound    completely or near-completely within the polymeric shell and to    produce nanoparticles of the drug or compound having a particle size    of 30 to 150 μm. Such a perfusion fluid remains stable for more than    24 hours with more 95% drug remaining loaded in the polymeric    micelles.

It should be noted herein that the selection of the diluting fluid,largely depends on the nature of the poorly water-soluble drug orcompound utilized as well as on the disorder for which thepharmaceutical composition is administrated. Suitable diluting fluidsmay be selected from, but not limited to water, saline, dextrose 5% and10% solutions, dextrose and sodium chloride solution, sodium lactatesolution, lactated Ringer solution, mannitol solution, mannitol withdextrose or sodium chloride solution, Ringer's solution, sodium chloridesolution, sterile water for injection and multiple electrolyte solutionscomprising varying combinations of electrolytes, dextrose, fructose andinvert sugar. Preferably, the diluting fluid is a fluid comprisingdextrose and water and more preferably dextrose 5% and 10% solutions.

The preferred method for preparation of the nanoparticulatepharmaceutical composition of the present invention and itsadministration to patients in need thereof is graphically represented inFIG. 11.

The invention is further described in detail with respect to thefollowing non-limiting examples, which, however, should in no way beconstrued as limiting the scope of the invention.

It should be noted that in the Examples set forth hereinbelow, thediafiltration equipment utilized for purification of the polymer was aProflux M12 diafiltration device (Make: Millipore) and the dialysisequipment used for purification of the polymer was of Cellulosemembrane-D-9402 (Make: Sigma)

Experimental Section Reference Example-1 Preparation of the PolymerUsing the Dialysis Method

The polymerization reaction was carried out in a 2 L glass vessel. 24 gof N-Isopropyl acrylamide, 12 ml of distilled 1-Vinyl-2-pyrrolidone and4 g of Polyethylene glycol (mol. wt 6000) ester of Maleic anhydride(MPEG) were added to about 2 L of water. To this 11.2 ml of aqueousN,N′-Methylenebisacrylamide (MBA) solution [49 mg/ml] and 8 ml ofTetramethylethylenediamine (d=0.77 gm/ml) were added. The solution wasde-aerated by bubbling nitrogen gas for 30 minutes. Then 8 ml of aqueousFerrous ammonium Sulphate (0.5% w/v) and 12 ml of aqueous AmmoniumPersulphate (80% w/v) were added and the reaction was continued for 3hours with continuous nitrogen bubbling. Polymerization was carried outat 34° C. in a water bath with shaking at 80 rpm.

The solution was filled in dialysis bags and was descended in water(dialysis medium). Dialysis was carried out for 24 hours changing wateronce. After 24 hours, the solution was removed from the dialysis bagsand lyophilized in round bottom flasks.

The detection and quantification of the residual monomers, especiallyresidual VP and NIPAM in the polymer were carried out by Agilent 1100series HPLC system, using Reverse Phase RP-18 (C-18) columns[Lichrospher RP-18e, 5μ, 250 mm×4 mm]. The Mobile Phase used was amixture of water and acetonitrile in a ratio of 80:20, at a flow rate of1 ml/min, with a sample injection volume was 50 μl. The run time was 10mins and the column temperature was 30° C. and the Detector wavelengthwas 226 nm. Under the above conditions, NIPAM had a retention time ofabout 3 minutes, whereas VP had a retention time of about 5 minutes.

Analytical Data: % Residual Monomers i) NIPAM=0.066% (660 ppm) and ii)VP=0.011% (110 ppm).

Example-1 Preparation of the Polymer Using the Diafiltration Method

The polymerization reaction is carried out in two 5 L glass vessels fora batch size of 160 g (4 L×2) of the polymer. To each vessel, 48 g ofN-Isopropylacrylamide, 23 ml of distilled 1 Vinyl-2-pyrrolidone and 8 gof Polyethylene glycol (mol. wt 6000) ester of Maleic anhydride (MPEG)were added to about 4 L of water. To this 22.4 ml of aqueousN,N′-Methylenebisacrylamide (MBA) solution [49 mg/ml] and 16 ml ofTetramethylethylenediamine were added. The solution was de-aerated bybubbling nitrogen gas for 30 minutes. Then 16 ml of aqueous Ferrousammonium Sulphate (0.5% w/v) and 24 ml of aqueous Ammonium Persulphate(80% w/v) were added and the reaction was continued for 3 hours withcontinuous nitrogen bubbling. Polymerization was carried out at 34° C.in a water bath with shaking at 80 rpm. During polymerization, sampleswere withdrawn at appropriate time points (0, 15, 60 and 180 minutes)for reaction monitoring.

After polymerization was complete, the solution was filtered throughpre-sterilized, disposable 0.2 μm Polyethersulphone membrane 1″ capsulefilters of 0.8 and 0.2 μm pore size; Type DPS-5101AA-201, mfg byAdvanced Microdevices Pvt. Ltd, India). The filtered contents of boththe reaction vessels were pooled and subjected to tangential flowfiltration using Proflux M12 (Millipore) diafiltration device to removeresidual monomers and other low molecular weight impurities. Thecombined lot of 8 L of the reaction mixture was initially concentratedto around 2.2 L through diafiltration and then the resultant concentrateis diafiltered using around 30 L of highly purified water. Duringdiafiltration, the reaction mixture is concentrated to around 1 L. Totalprocessing time for the diafiltration for a batch size of 160 g (8L) isaround 4-6 hours. Diafiltered solution is then subjected tolyophilization.

The detection and quantification of the residual monomers, especiallyresidual VP and NIPAM in the polymer were carried out by Agilent 1100series HPLC system, using Reverse Phase RP-18 (C-18) columns[Lichrospher RP-18e, 5μ, 250 mm×4 mm]. The Mobile Phase used was amixture of water and acetonitrile in a ratio of 80:20, at a flow rate of1 ml/min, with a sample injection volume was 50 μl. The run time was 10mins and the column temperature was 30° C. and the Detector wavelengthwas 226 nm. Under the above conditions, NIPAM had a retention time ofabout 3 minutes, whereas VP had a retention time of about 5 minutes.

Analytical Data: % Residual Monomers i) NIPAM=<0.001% (<10 ppm) and ii)VP=<0.001% (<10 ppm)

The polymer had the following spectral characteristics, viz.

¹H NMR (300 MHz, Bruker Spectometer, CDCl₃, δ ppm): 1.15 (br,—CH(CH₃)₂); 1.45 (br, —CH₂—CH—N(VP-Ring); 1.63 (br, —CH₂—CHC(═O)NH);1.99 (br, —CH C(═O)NH—), CH₂ (VP ring), 2.36 (CH₂, VP ring), 3.0(—O—CH₂—CH₂—), 3.23 (CH₂—N—); 3.62-3.66 (Br, CH₂, MPEG); 3.72(NH—CH(CH₃)₂); 3.97 (Br, CH)

¹³C NMR (300 MHz, Bruker Spectrometer, CDCl₃, δ ppm): 174 (C═O);76.6-77.6 (multiplet for CDCl₃ and CH for polymer backbone), 70.6 (CH₂'sMPEG), 41.6 (CH for isopropyl unit), 31.8 (CH₂ 's, polymer backbone),22.6 (CH₃ 's, isopropyl)

FTIR (KBr Pellet, cm⁻¹): 3500 (s, OH); 3296 (s, NH, sec-Amide),2972-2933 (s, CH, CH₂, CH₃), 1546 (s, NH bend of Amide II and possiblyC═O of free acid, minor), 1387, 1367 (doublet of Isopropyl groups, CH₃,deformation), 1172-1129 (m, O—C—O)

Example-2 Preparation of Paclitaxel Nanoparticulate PharmaceuticalComposition (Reconstitution in Small Volume i.e. Up to 40 ml)

A] Preparation of Alcoholic solution of Paclitaxel (20 mg/ml): 200 mg ofPaclitaxel was dissolved in 10.0 ml of Ethanol.B] Preparation of Aqueous Concentrate of Polymer and Excipients: 100 mgof the Polymer obtained by Example-1, 66.7 mg of sodium deoxycholate and100 mg of sodium citrate were dissolved in 10 ml water to give a clearsolution.C] Preparation of Paclitaxel Nanoparticles (0.6 mg/ml): 1.0 ml of theaqueous concentrate of the polymer and excipients of step B] wasdissolved in 31.3 ml of 10% Dextrose solution to obtain a clearsolution. 1.0 ml of the alcoholic solution of Paclitaxel of step A] wasadded to the above solution through needle having an internal diameterof 0.330 mm within 4 seconds to obtain nanoparticles of Paclitaxel at aconcentration of 0.6 mg/mlThe pharmaceutical composition thus prepared had the followingcharacteristics:

Polymer Sodium Paclitaxel Particle Concn. deoxycholate Sodium CitrateConcn. Size Dilution Fluid (mg/ml) Concn. (mg/ml) Concn. (mg/ml) (mg/ml)(nm) Stability 10% Dextrose 0.3 0.2 0.3 0.6 ≈80 >24 hrs

Example-3 Preparation of Paclitaxel Nanoparticulate PharmaceuticalComposition (Reconstitution in Large Volume i.e. Up to 500 ml)

A] Preparation of Alcoholic solution of Paclitaxel (20 mg/ml): 400 mg ofPaclitaxel was dissolved in 20.0 ml of Ethanol.B] Preparation of Aqueous Concentrate of Polymer and Excipients: 200 mgof the Polymer obtained by Example-1, 133.4 mg of sodium deoxycholateand 200 mg of sodium citrate were dissolved in 20 ml water to give aclear solution.C] Preparation of Paclitaxel Nanoparticles (0.6 mg/ml): 15.0 ml of theaqueous concentrate of the polymer and excipients of step B] wasdissolved in 500 ml of 10% Dextrose solution to obtain a clear solution.15.0 ml of the alcoholic solution of Paclitaxel of step A] was added tothe above solution through a needle having an internal diameter of 0.711mm within 8 seconds to obtain nanoparticles of Paclitaxel at aconcentration of 0.6 mg/mlThe pharmaceutical composition thus prepared had the followingcharacteristics:

Polymer Sodium Paclitaxel Particle Concn. deoxycholate Sodium CitrateConcn. Size Dilution Fluid (mg/ml) Concn. (mg/ml) Concn. (mg/ml) (mg/ml)(nm) Stability 10% Dextrose 0.3 0.2 0.3 0.6 ≈85 >24 hrs

Example-4 Preparation of Nanoparticulate Pharmaceutical Composition of aBetulinic Acid Derivative [MJ-1098 of Formula (II)]

A] Preparation of a solution of MJ-1098 (15 mg/ml): MJ-1098 (15 mg) wasdissolved in a mixture of 0.15 ml of N,N-Dimethylacetamide, 0.01 ml ofPolysorbate 80 and 0.84 ml of Ethanol was added to the above solutionand dissolved by sonication.B] Preparation of Aqueous Concentrate of Polymer and Excipients: 10 mgof the Polymer obtained by Example-1, 6.67 mg of sodium deoxycholate and10 mg of sodium citrate were dissolved in 1 ml water to give a clearsolution.C] Preparation of MJ-1098 Nanoparticles (0.75 mg/ml): 0.3 ml of theaqueous concentrate of the polymer and excipients of step B] wasdissolved in 9.2 ml of 5% Dextrose solution to obtain a clear solution.0.5 ml of the solution of MJ-1098 of step A] was added to the abovesolution through a needle having an internal diameter of 0.330 mm within3 seconds to obtain nanoparticles of MJ-1098 at a concentration of 0.75mg/mlThe pharmaceutical composition thus prepared had the followingcharacteristics:

Polymer Sodium Mj-1098 Particle Concn. deoxycholate Sodium CitrateConcn. Size Dilution Fluid (mg/ml) Concn. (mg/ml) Concn. (mg/ml) (mg/ml)(nm) Stability 5% Dextrose 0.3 0.2 0.3 0.75 ≈62 >24 hrs

Example-5 Preparation of Nanoparticulate Pharmaceutical Composition of aBetulinic Acid Derivative [DRF-4012 of Formula (III)]

A] Preparation of a solution of DRF-4012 (20 mg/ml): MJ-DRF-4012 (20 mg)was dissolved in a mixture of 0.01 ml of Polysorbate 80 and 0.99 ml ofEthanol and dissolved by sonication.B] Preparation of Aqueous Concentrate of Polymer and Excipients: 10 mgof the Polymer obtained by Example-1, 6.67 mg of sodium deoxycholate and10 mg of sodium citrate were dissolved in 1 ml water to give a clearsolution.C] Preparation of DRF-4012 Nanoparticles (0.60 mg/ml): 0.33 ml of theaqueous concentrate of the polymer and excipients of step B] wasdissolved in 10.44 ml of 5% Dextrose solution to obtain a clearsolution. 0.33 ml of the solution of DRF-4012 of step A] was added tothe above solution through a needle having an internal diameter of 0.305mm within 3 seconds to obtain nanoparticles of DRF-4012 at aconcentration of 0.6 mg/mlThe pharmaceutical composition thus prepared had the followingcharacteristics:

Polymer Sodium DRF-4012 Particle Concn. deoxycholate Sodium CitrateConcn. Size Dilution Fluid (mg/ml) Concn. (mg/ml) Concn. (mg/ml) (mg/ml)(nm) Stability 5% Dextrose 0.3 0.2 0.3 0.6 ≈70 >24 hrs

Example-6 Preparation of Nanoparticulate Pharmaceutical Composition of aBetulinic Acid Derivative [DRF-4015 of Formula (IV)]

A] Preparation of a solution of DRF-4015 (20 mg/ml): MJ-DRF-4015 (20 mg)was dissolved in a mixture of 0.01 ml of Polysorbate 80 and 0.99 ml ofEthanol and dissolved by sonication.B] Preparation of Aqueous Concentrate of Polymer and Excipients: 10 mgof the Polymer obtained by Example-1, 6.67 mg of sodium deoxycholate and10 mg of sodium citrate were dissolved in 1 ml water to give a clearsolution.C] Preparation of DRF-4015 Nanoparticles (0.60 mg/ml): 0.33 ml of theaqueous concentrate of the polymer and excipients of step B] wasdissolved in 10.44 ml of 5% Dextrose solution to obtain a clearsolution. 0.33 ml of the solution of DRF-4015 of step A] was added tothe above solution through a needle having an internal diameter of 0.330mm within 4 seconds to obtain nanoparticles of DRF-4015 at aconcentration of 0.6 mg/mlThe pharmaceutical composition thus prepared had the followingcharacteristics:

Polymer Sodium DRF-4015 Particle Concn. deoxycholate Sodium CitrateConcn. Size Dilution Fluid (mg/ml) Concn. (mg/ml) Concn. (mg/ml) (mg/ml)(nm) Stability 5% Dextrose 0.3 0.2 0.3 0.6 ≈46 >24 hrs

Example-7 Preparation of Docetaxel Nanoparticulate PharmaceuticalComposition

A] Preparation of Alcoholic solution of Docetaxel (40 mg/ml): 200 mg ofDocetaxel was dissolved in 5.0 ml of Ethanol.B] Preparation of Aqueous Concentrate of Polymer and Excipients: 400 mgof the Polymer obtained by Example-1,400 mg of sodium deoxycholate and400 mg of sodium citrate were dissolved in 10 ml water to give a clearsolution.C] Preparation of Docetaxel Nanoparticles (0.5 mg/ml): 4.0 ml of theaqueous concentrate of the polymer and excipients of step B] wasdissolved in 35.5 ml of 10% Dextrose solution to obtain a clearsolution. 0.5 ml of the alcoholic solution of Docetaxel of step A] wasadded to the above solution through a needle having an internal diameterof 0.330 mm within 3 seconds to obtain nanoparticles of Docetaxel at aconcentration of 0.5 mg/mlThe pharmaceutical composition thus prepared had the followingcharacteristics:

Polymer Sodium Docetaxel Particle Concn. deoxycholate Sodium CitrateConcn. Size Dilution Fluid (mg/ml) Concn. (mg/ml) Concn. (mg/ml) (mg/ml)(nm) Stability 10% Dextrose 4.0 4.0 4.0 0.5 ≈125 >24 hrs

Example-8 Preparation of Etoposide Nanoparticulate PharmaceuticalComposition

A] Preparation of a solution of Etoposide (20 mg/ml): 20 mg of Etoposidewas dissolved in a mixture of 0.10 ml of N,N-dimethyl acetamide and 0.90ml of Ethanol under sonication.B] Preparation of Aqueous Concentrate of Polymer and Excipients: 10 mgof the Polymer obtained by Example-1, 6.67 mg of sodium deoxycholate and10 mg of sodium citrate were dissolved in 10 ml water to give a clearsolution.C] Preparation of Etoposide Nanoparticles (0.6 mg/ml): 0.3 ml of theaqueous concentrate of the polymer and excipients of step B] wasdissolved in 9.4 ml of 5% Dextrose solution to obtain a clear solution.0.3 ml of the alcoholic solution of Etoposide of step A] was added tothe above solution through a needle having an internal diameter of 0.330mm within 3 seconds to obtain nanoparticles of Etoposide at aconcentration of 0.6 mg/mlThe pharmaceutical composition thus prepared had the followingcharacteristics:

Polymer Sodium Etoposide Particle Concn. deoxycholate Sodium CitrateConcn. Size Dilution Fluid (mg/ml) Concn. (mg/ml) Concn. (mg/ml) (mg/ml)(nm) Stability 5% Dextrose 0.3 0.2 0.3 0.6 ≈50 >24 hrs

Example-9 Determination of Pharmacokinetics, Biodistribution, andElimination of [¹⁴C]-Labelled Polymer in Mice

30 male Swiss albino mice, 6-8 weeks of age, weighing approximately25-30 gms, were randomly divided into five groups consisting of sixanimals each. [¹⁴C]-labelled polymer was diluted in deionised water to 5mg/ml based on the specific activity of the polymer. All animalsreceived a single dose of [¹⁴C] polymer 40 mg/kg by intravenousinjection.

In the pharmacokinetics study 100 μl of blood was collected from theanimals by retro-orbital bleeding under anesthesia at time points of 3,10, 30 min, 1, 2, 4, 8, 16, and 24 hrs post administration into EDTAcontaining tubes. For excretion studies, urine and feaces was collectedfrom the metabolic cage or by force (10 minutes). At termination (10min, 60 min, 24 and 48 hrs) adrenal, brain, lungs, liver, heart,kidneys, spleen, stomach, small intestine, large intestine, feaces,urine, urinary bladder, eye, skin, skin, thigh muscle, testing andepididymis were collected, rinsed, excised and weighed.

The concentrations of the [¹⁴C] polymer in blood and urine weredetermined by combining 50 μl of blood/urine with 5 ml of liquidscintillation cocktail. Faeces and tissues (not more than 0.5 gms) werehomogenized in deionised water to obtain 20% homogenate before combining500 μl with 5 ml of liquid scintillation cocktail. Samples were analysedby liquid scintillation analyser. The counts per minute (CPM) wereconverted to amount of the [¹⁴C] polymer in μg/ml based on linearity andQuenching curves.

The radioactive blood concentration profile revealed a bi-phasic curvewith short elimination half-life T_(1/2) (β) of 0.448±0.157 hours (26.88min) and rapid clearance of 54.7 ml/hr.

The dominant route of elimination was found to be urine (urine, 66.91%vs feaces, 17.39% at 48 hrs) and recovery data collected up to 48 hrsaccounts for 84.87% of radioactivity injected. Tissue distribution wasnegligible. The kidney, liver, skin and intestine were found to be thetarget organs. However, the level of the polymer in tissues was rapidlycleared via urine and faeces.

Tissue distribution was negligible in kidney, liver, skin and intestinepresenting with the highest levels of radioactivity. However, levels ofpolymer in tissues were rapidly cleared via urine and feaces.

In conclusion, the study shows that the polymer is rapidly eliminatedfrom the body without being deposited in vital organs. Although, thepolymer is known to be non-biodegradable, the rapid and efficientclearance primarily via urine suggests the safety and utility of thepolymer for human use.

Example-10 Determination of the Possible Local Toxicity, if any, at theSite of Administration Upon Five Day Intravenous Bolus Administration of125 mg/kg of the Polymer in Rabbits

The test substance dissolved in dextrose 10% at a concentration of 75mg/ml was administered intravenously with a 5 ml disposable syringe and23G needle into the marginal vein of the right ear of each rabbit at 125mg/kg daily for five consecutive days. Left ear served, as control andreceived 10% Dextrose by the same route. Dosing volume was adjusted tonot more than 3.5 ml/Kg. Body weight of animal. Periodic observationsfor local toxicity were made at the site of injection at 5 min, 10 min,30 min, 60 min and 24 hrs on each day for days 1 to 5. Punch biopsies atsite of injection were taken from both ears of all six rabbits on Day 7.

A five day continuous intravenous administration of 75 mg/ml of thepolymer at a dose of 125 mg/kg in rabbit ear vein causes mild tomoderate thrombophlebitis at the site of injection of 10% Dextroseinjected rabbits. It may be concluded that the dose of the polymerselected does not cause any local toxicity at the site ofadministration.

Example-11 Determination of the Possible Target Organ(s) of Toxicitywith Special Reference to Microvasculature Upon Five Days IntravenousBolous Administration of 400 mg/kg of the Polymer in Wistar Rats

The test substance was dissolved in Dextrose 10% and administeredintravenously with the help of a 5 ml disposable syringe and 23 G needleinto the tail vein of each rat at 400 mg/kg. Control animals received 05Dextrose only by the same route. Dosing volume was adjusted to 5 ml/kgbody weight of the animals. Periodic observations (Day 7,14 and 21post-treatment) on adverse effects (general examination and laboratoryparameters) and deaths were recorded. All the animals were sacrificedand necropsied.

Under the conditions of study, five-day intravenous bolus administrationof the polymer at a dose level of 400 mg/kg body weight does not produceany mortality or any physical toxic signs or symptoms in treated rats.

Individual and mean body weights of rats showed a steady increase inboth the polymer treated and control groups. No significant differencewas noted for body weight for treated animals as compared to that of thecontrol.

In rats treated with the polymer, haematological parameters were withinnormal limits throughout the study. Significant differences weredetected at base line for total bilirubin (p=0.0471) and Uric acid(p=0.0157) for interim group and total protein (p=0.0005) and Uric acid(p=0.0404) for terminal group animals over the control animals. However,all values are within the normal limits.

The photoactometer test showed that there was no significant differencebetween the locomotor activity between the control and treated groups onday 7 and 21 respectively suggesting that the polymer does not have anyneurotoxicity.

The treated and control groups specimens showed similar histologicalfeatures. All organs studied showed normal structure on lightmicroscopic examination. The microvasculature in each organ wascarefully examined and no pathological features were seen in any of theorgans. Further, there were no changes in microvasculature of thepolymer treated animals.

From the above observations, it was seen that the polymer at a dose of400 mg/kg body weight administered for five consecutive days did notcause any general toxicity or any significant haematological toxicity.However, total bilirubin was found to be significantly higher for theterminal group as compared top the control group on Day 21.

Example-12 Determination of the Possible Target Organ(s) of Toxicitywith Special References to Microvasculature Upon Single IntravenousBolus Administration of 800 mg/kg of the Polymer in Wistar Rats

The test substance dissolved in dextrose 10% was dissolved in Dextrose10% was administered with the help of a 1 ml disposable syringe and 26 Gneedle into the tail vein of each rat at 800 mg/kg. Control animalsreceived 10% dextrose only by the same route. Dosing volume was adjustedto 5 ml/kg body weight of the animals. Periodic observations (Day 1, 3and 7 post-treatment) on adverse effects (general examination andlaboratory parameters) and deaths were recorded. All the animals weresacrificed and necropsied.

Under the conditions of the study, single intravenous bolusadministration of the polymer at a dose level of 800 mg/kg Body weightdoes not produce any mortality or any observable toxic signs or symptomsin rats.

Individual and mean body weights of rats show a steady increase in boththe polymer treated and control groups. In rats treated with thepolymer, haematological parameters were within normal limits throughoutthe study. In rats treated with the polymer, biochemical parameters werewithin normal limits throughout the study. The histopathological studiesshow that there is no significant difference between control and treatedgroups of rats. Photomicrographs of the polymer treated rats sacrificedon Day 3 and Day 7 post injection show that there are no apparentmicrovasculature changes in all four organs examined (Brain, Eye, Kidneyand Skin)

From the above observations it is seen that the polymer at a dose of 800mg/kg body weight does not cause any general toxicity or any significanthaematological and biochemical toxicity or changes in microvasculatureand can be considered to be safe when administered intravenously inrats.

Example-13 Determination of the Possible Local Toxicity, if any, at theSite of Administration (Subcutaneous) of the Polymer in Rabbits

A single injection of 0.1 ml of the test substance dissolved in dextrose10% at a concentration of 75 mg/ml was administered subcutaneously with1 ml disposable syringe and 23G needle into the right ear lobe of eachof the six rabbits. Control will receive 0.1 ml of 10% dextrose by thesame route in the left ear lobe of all six rabbits. Periodicobservations for local toxicity were made at the site of injection at 5min, 10 min, 30 min, 60 min and 24 hrs.

A single sub cutaneous administration of 100 μl of 75 mg/ml of thepolymer or 100 μl of Dextrose in the rabbit ear causes mild inflammationat the site of injection when tested after 48 hrs post injection. It maybe concluded that the polymer selected does not cause any local toxicityat the site of administration following sub-cutaneous administration

Example-14 Determination of Six Months Dose Toxicity Study byIntravenous Route of the Polymer in Rats

The polymer used in nanoparticle formulation was administered at doselevel equivalent to 10 mg/kg of drug. Controls were administeredDextrose (10%) intravenously in the lateral tail vein cyclically onceevery three weeks for a period of 180 Days (approximately 26 weeks).Observations comprised of mortality, clinical signs, body weight, foodand water consumption, clinical laboratory investigations, organ weightsand histopathology.

Animals of treated and control groups remained generally active andhealthy during the period of study. There was no treatment relatedmortality except few incidental deaths due to infections in bothtreatment and control groups. Animals of both sexes showed a progressiveincrease in body weight and there were no changes in feed or waterconsumption during the study. Haematology parameters in both males andfemales were within the normal range as reported for Wistar rats.However, in the treatment group, there was minor decrease, yet withinnormal limits, of WBC and neutrophile counts in males and Neutrophilecount in females. A mild increase in Reticulocyte count was noticed inboth treated and control group.

Blood biochemistry parameters in both males and females were within thenormal range as reported for Wistar rats. Minor Changes includedslightly higher than normal values in Glucose, ALP and Creatinine inboth males and females of treated and control groups. Mild increase inTriglycerides of treatment group males was seen at 6 months. Urineparameters in both males and females were within normal limits.

1. A polymer comprising of three monomeric units, selected from1-Vinylpyrrolidone (VP), N-Isopropylacrylamide (NIPAM), and ester ofMaleic anhydride and Polyethylene glycol (MPEG), cross-linked with abi-functional vinyl derivative, of high purity and substantially free ofrespective toxic monomeric contaminants.
 2. A polymer comprising ofthree monomeric units, selected from 1-Vinylpyrrolidone (VP),N-Isopropylacrylamide (NIPAM), and ester of Maleic anhydride andPolyethylene glycol (MPEG), cross-linked with a bi-functional vinylderivative, of high purity, containing respective toxic monomericcontaminants in amounts less than 0.001%.
 3. A polymer of high purityaccording to claim 1, wherein the bi-functional vinyl cross-linkingagent is N,N′-Methylene bis acrylamide (MBA).
 4. A polymer of highpurity according to claim 1, containing toxic 1-Vinylpyrrolidone (VP) inamounts less than 0.001%.
 5. A polymer of high purity according to claim1, containing toxic N-Isopropylacrylamide (NIPAM) in amounts less than0.001%.
 6. A polymer according to claim 1, wherein the weight ratio ofthe monomers, NIPAM:VP is in the range of between 55:22 to 65:35.
 7. Apolymer according to claim 6, wherein the weight ratio of the monomers,NIPAM:VP is in the range of between 58:32 to 62:28.
 8. A polymeraccording to claim 1, wherein the weight ratio of monomers,(NIPAM+VP):MPEG is in the range of between 90:10 to 95:5.
 9. A polymeraccording to claim 8, wherein the weight ratio of monomers,(NIPAM+VP):MPEG is in the range of between 80:20 to 95:5.
 10. A polymeraccording to claim 1, having peaks of δ at 174, 76.6-77.6, 70.6, 41.6,31.8, and 22.6 in their ¹³C NMR spectrum.
 11. A polymer according toclaim 1, having peaks of 6 at 1.14, 1.45, 1.63, 1.99, 2.36, 3.0, 3.23,3.62-3.66, 3.72, and 3.97 in their ¹H NMR spectrum.
 12. A polymeraccording to claim 1, having frequency of values cm⁻¹ at as 3500, 3296,2972-2933, 1546, 1387, 1367, and 1172-1129 in their FT-IR spectrum. 13.A polymer according to claim 1, having structure of formula (I),


14. A polymer according to claim 1, which is biocompatible.
 15. Apolymer according to claim 1, which is non-biodegradable.
 16. A polymeraccording to claim 1, which is non-toxic.
 17. A polymer according toclaim 1, having a T_(1/2) (K10) value of 0.152±0.018 hours.
 18. Apolymer according to claim 1, having T_(1/2) (α) value of 0.065±0.014hours.
 19. A polymer according to claim 1, having a T_(1/2) (β) value of0.448±0.0157 hours.
 20. A polymer according to claim 1, having a C_(max)value of 82.96±5.11 μg/ml.
 21. A polymer according to claim 1, having anArea Under the Curve (AUC) value of 18.29±1.62 hours×μg/ml.
 22. Apolymer according to claim 1, having a Clearance Time (CL) value of54.67±4.86 ml/hr.
 23. A polymer according to claim 1, having a MeanResidence Time (MRT) value of 0.465±0.13 hours.
 24. A polymer accordingto claim 1, having a Volume Distribution Under Steady State (V_(ss))value of 25.43±5.2 ml/hr.
 25. A polymer according to claim 1, which iseliminated from urine, faeces, tissues, and rinse.
 26. A polymeraccording to claim 1, which is predominantly eliminated from urine andfaeces.
 27. A polymer according to claim 1, about 67% of which iseliminated from urine 48 hours post dosing.
 28. A polymer according toclaim 1, about 17% of which is eliminated from faeces 48 hours postdosing.
 29. A polymer according to anyone of clams claim 1, about 84% ofwhich is eliminated from urine, faeces, tissues and rinse, 48 hours postdosing.
 30. A polymer according to claim 1, which does not cause anylocal toxicity at the site of administration, 48 hours post subcutaneousadministration of an aqueous solution of the said polymer in a rabbitear.
 31. A polymer according to claim 1, which does not cause any localtoxicity at the site of administration, 24 hours post intravenousadministration of an aqueous solution of the said polymer in a rabbitear vein.
 32. A polymer according to claim 1, which does not cause anygeneral toxicity, when administrated through an intravenous bolar routeto Wistar rats up to five consecutive days at a dose of between 400mg/kg to 800 mg/kg.
 33. A polymer according to claim 1, which does notcause any significant haematological toxicity, when administratedthrough an intravenous bolar route to Wistar rats up to five consecutivedays at a dose of between 400 mg/kg to 800 mg/kg.
 34. A [¹⁴C]-labelledpolymer comprising of three monomeric units, selected from1-Vinylpyrrolidone (VP), N-Isopropylacrylamide (NIPAM), and ester ofMaleic anhydride and Polyethylene glycol (MPEG), cross-lined withN,N′-Methylene bis acrylamide (MBA).
 35. A [¹⁴C]-labelled polymercomprising of three monomeric units, selected from 1-Vinylpyrrolidone(VP), N-Isopropylacrylamide (NIPAM), and ester of Maleic anhydride andPolyethylene glycol (MPEG), cross-lined with N,N′-Methylene bisacrylamide (MBA).
 36. A [¹⁴C]-labelled polymer of high purity accordingto claim 34, wherein the weight ratio of the monomers, NIPAM:VP is inthe range of between 55:22 to 65:35, and the ratio of monomers,(NIPAM+VP):MPEG is in the range of between 90:10 to 95:5.
 37. A[¹⁴C]-labelled polymer of high purity according to claim 34, wherein theweight ratio of the monomers, NIPAM:VP is in the range of between 58:32to 62:28, and the ratio of monomers, (NIPAM+VP):MPEG is in the range ofbetween 80:20 to 95:5. 38-107. (canceled)